|Year : 2022 | Volume
| Issue : 1 | Page : 21-29
Osteogenic differentiation from mouse adipose-derived stem cells and bone marrow stem cells
Cheng-Pu Huang1, Keng-Chia Hsu1, Chean-Ping Wu2, Hsi-Tien Wu1
1 Department of BioAgricultural Sciences, College of Agriculture, National Chiayi University, Chiayi, Taiwan
2 Department of Animal Science, College of Agriculture, National Chiayi University, Chiayi, Taiwan
|Date of Submission||13-Jul-2021|
|Date of Decision||21-Nov-2021|
|Date of Acceptance||14-Dec-2021|
|Date of Web Publication||25-Feb-2022|
Dr. Hsi-Tien Wu
Department of BioAgricultural Sciences, College of Agriculture, National Chiayi University, 300 Syuefu Road, Chiayi 600355
Source of Support: None, Conflict of Interest: None
Mesenchymal stem cells (MSCs) have been successfully cultured and proliferated in vitro and can differentiate into a variety of specific cell types, such as adipocytes or osteocytes, through chemical stimulation. One of the major applications of MSCs is in regenerative medicine research. MSCs can be collected from many adult tissues. In this experiment, an 8-week-old expresses green fluorescent protein (EGFP) transgenic mouse, FVB/NCrl-Tg(Pgk1-EGFP)01Narl, was used to obtain adipose-derived stem cells (ADSCs) from abdominal adipose tissue and bone marrow stem cells (BMSCs) from femur bone marrow. We compared the differences in the growth rate and differentiation ability of ADSCs and BMSCs. The growth curves of different generations (P1 and P3) of the stem cells showed that the proliferation rate of ADSCs was significantly higher than that of BMSCs. The purity of stem cells was measured by the number of colony-forming unit fibroblast. The results show that the number of colonies of ADSCs at different generations (P1 and P3) was significantly higher than that of BMSCs and that the purity of ADSCs was greater than that of BMSCs. Comparing the ability of ADSCs and BMSCs to induce osteogenic differentiation and the expression of Runx2 and Opn genes, the results show that ADSCs had a higher rate of osteogenic differentiation than BMSCs. In summary, mouse ADSCs display similar osteogenic differentiation ability to BMSCs but have a better capacity than BMSCs in terms of stem cell purity and cell proliferation in vitro.
Keywords: Adipogenic differentiation, adipose-derived stem cells, bone marrow stem cells, mesenchymal stem cells, osteogenic differentiation
|How to cite this article:|
Huang CP, Hsu KC, Wu CP, Wu HT. Osteogenic differentiation from mouse adipose-derived stem cells and bone marrow stem cells. Chin J Physiol 2022;65:21-9
| Introduction|| |
Mesenchymal stem cells (MSCs) are a nonhematopoietic series of adherent and multilineage stem cells with the ability to self-renew and differentiate into a variety of cell types, including adipocytes, chondrocytes, myocytes, and osteoblasts., MSCs have gradually been shown to play key roles in tissue healing and regenerative medicine. The differentiation potential and the paracrine properties of MSCs have made them a key option for tissue repair. At present, MSCs have been used in cell therapy, such as in treatments for myocardial damage and liver fibrosis.,,,
The development of regenerative medicine has enabled researchers to obtain a large number of MSCs in the body and stably culture them in vitro for expansion while retaining the ability to differentiate into a variety of tissues. Bone marrow-derived MSCs (BMSCs) are the most frequently used stem cells in cell therapy and tissue engineering. However, it has also been found that MSCs exist in other tissues, such as adipose, cartilage, and umbilical cord blood. Different sources of MSCs, such as adipose-derived stem cells (ADSCs) from adipose tissue and BMSCs from the bone marrow cavity, have slightly different stem cell characteristics, such as growth rates and differentiation potentials.
The in vitro collection, purification, identification, and expansion of primordial stem cells are the first step in the study of MSCs. Then, MSCs are cultured in a differentiation medium containing specific inducing factors to promote gene expression and cell differentiation into adipocytes, chondrocytes, or osteoblasts. MSCs can undergo an osteogenic differentiation pathway under specific induction conditions. Many regulatory factors participate in the initiation of the osteogenic differentiation pathway of MSCs, such as bone morphogenetic proteins and Wnt signaling molecules. Many genes are related and processed to the osteogenic differentiation pathway, such as Tgf-β, Runx 2, Alp, Col1a1, Opn, and Ocn. In this study, we focused on the ability of mouse MSCs to differentiate from ADSCs and BMSCs into osteoblasts.
The differentiation of MSCs into osteoblasts can be divided into four steps: cell proliferation, extracellular matrix production, osteoblast maturation, and osteoblast mineralization. When stem cells are not stimulated by specific differentiation induction factors, they mainly undergo self-renewal and cell proliferation. When stem cells are stimulated by osteogenic differentiation factors, they slow down cell proliferation and turn on early osteogenic differentiation genes. For example, the protein-coding gene Runx2 can turn on a variety of downstream-related genes in osteogenic differentiation. In the step of extracellular matrix production, stem cells differentiate into osteoprogenitor cells, a large number of intercellular substances are produced, and the cells are closely connected. Osteoprogenitor cells express genes such as collagen Type I (Col1a1) and alkaline phosphorylase (Alp). COL1A1 is the main component of MSCs; ALP functions to dephosphorylate in an alkaline environment in the cell. During osteoblast maturation, stem cells absorb calcium in the late stage of osteogenic differentiation and express genes such as osteopontin (Opn) and osteocalcin (Ocn). OPN is a phosphorylated glycoprotein often found in mineralized interstitial cells of the body. OPN can bind to a variety of proteins and make the extracellular matrix structure more compact. OCN can interact with hydroxyapatite, bind inorganic substances together, and fix calcium ions as important proteins for mineralization. The last step of the differentiation of MSCs into osteoblasts is the osteoblast mineralization of intercellular substances and the maturation of osteocytes.
The purpose of the present study was to compare the characteristics of mouse ADSCs and BMSCs, including MSC surface antigens, MSC growth rate, number of fibroblast colony-forming units (CFU), and adipose differentiation and osteogenic differentiation abilities. Through osteogenic differentiation stimulation, we observed and analyzed the ability of osteoblasts to transform ADSCs and BMSCs and compared their differences. The results from this study can be used as reference for subsequent MSC research applications, such as the study of an osteoporosis repair model.
| Materials and Methods|| |
BMSCs and ADSCs were obtained from 5 FVB/NCrl-Tg(Pgk1-EGFP)01Narl mice that were 8 weeks old (National Laboratory Animal Center, Taiwan). The animals were kept in a clean conventional animal room with air, temperature, and light control. All animals were maintained, handled, and treated following NRC (National Research Council, USA) guidelines, and all experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at National Chiayi University, Taiwan (IACUC no. 103009).
Primary culture of mesenchymal stem cells
Bone marrow stem cells
Isolated and purified mouse BMSCs were cultured with frequent changes of the culture medium within the first 72 h. Briefly, after sacrificing the mice, the hind bones (femur and tibia) were cut with small holes on both sides, and a 27G syringe was used to wash and flush out the bone marrow cells from the bone marrow cavity with MEM-alpha (Mediatech, Manassas, VA, USA) containing 15% fetal bovine serum and 1% penicillin/streptomycin. The cells were filtered with a 70 μm cell strainer (BD Falcon, Sparks, MD, USA) and centrifuged at 450 g at 4°C for 5 min. After removing the supernatant and resuspending, the cells (2.5 × 107 cells/mL) were plated in 6-well culture dishes and then cultured at 37°C in a 5% CO2 incubator. After 3 h, 1 mL of the MEM-alpha was replaced. By using the attachment characteristics of BMSCs, the suspended nonattached cells were carefully removed, and 1 mL of fresh culture medium was replaced every 8 h within the first 72 h. After that, the fresh medium was replaced every 3 d until the cells reached 80% confluence, when subculture could be carried out.
Adipose derived stem cells
The method of collecting adipose-derived stem cells was modified according to Sugii et al. Fat tissue near the lower abdomen, gonads, and thighs of the mouse was collected. The fat tissue was thoroughly shred, and then 10 mL (0.05 mg/mL) of collagenase Type I solution (Thermo Fisher Scientific, Wilmington, DE, USA) was added. The sample was shaken and incubated for 30 min in a 37°C incubator. The disassociated cells were filtered through a 100 μm cell strainer (BD Falcon, Sparks, MD, USA). The collected cells were centrifuged and resuspended in an appropriate volume of MEM-alpha. A total of 6 × 106 cells per well were seeded in 6-well culture dishes and then cultured. The culture medium was replaced after 24 h, and the unattached cells were carefully removed. The medium was replaced every 3 d until the cells in the dish reached 80% confluence before subculturing.
Immunofluorescence staining detection of mouse bone marrow stem cells and ADSCs
The cells were cultured on a chamber slide (Nunc Lab-Tek II Chamber Slide System; Thermo Fisher Science). Cells were fixed with 4% paraformaldehyde, washed with DPBS, and blocked for 30 min at room temperature. CD44 (Invitrogen, Waltham, MA, USA), CD90 (BioLegend, San Diego, CA, USA), CD105 (BioLegend), and Sca-1 (BioLegend) fluorescent antibodies were separately resuspended in blocking buffer (100 μL) and incubated on the slide at 4°C in the dark for 1 h. After the antibody reaction, the slide was washed with DPBS, the chamber carrier was removed, and glass was mounted on the slide. Immunofluorescence detection was evaluated through observation under a laser confocal microscope (Leica TCS SP5; Leica Microsystems, Wetzlar, Germany).
Staining for differentiation markers of bone marrow stem cells and ADSCs
Bone marrow stem cell differentiation and staining
BMSCs were cultured in a 6-well plate and replaced with osteogenesis medium at 80% confluence. Fresh medium was changed every 3 d, and the cells were cultured for 14 d for staining. Osteogenesis medium based on MEM-alpha was supplemented with 50 μg/mL ascorbic acid 2-phosphate (Sigma–Aldrich), 10 nM dexamethasone (BioVision) and 10 mM β-glycerol phosphate (Sigma–Aldrich). After 14 d of osteogenic differentiation, BMSCs were washed with DPBS, and the cells were fixed with 4% paraformaldehyde in the dark. The cells were stained with 2% alizarin red S (VWR Life Science, Ontario, Canada) for 10 min until an orange precipitate appeared. After removing the excess dye, the cells were observed and recorded under a microscope. To quantify the percentage of cell mineralization, 10% cetylpyridinium chloride solution (Sigma–Aldrich) was added to the cells stained with alizarin red S. When the alizarin red S stain was completely dissolved, the OD value of the suspension was assessed at 570 nm.
ADSC differentiation and staining
After ADSCs reached 80% confluence, the medium was changed to adipogenesis medium. Adipogenesis medium based on MEM-alpha with an additional 50 μg/mL indomethacin (Sigma–Aldrich) and 100 nM dexamethasone (BioVision) was used to induce the differentiation of MSCs. The fresh medium was changed every 3 d, and the cells were cultured for 7 d for cell staining and RNA extraction. After 7 d of induced adipogenic differentiation, ADSCs were fixed with 4% paraformaldehyde in the dark and stained with Oil red O (Sigma–Aldrich) for 20 min. Excess dye was removed, and the cell morphology was observed and recorded under a microscope.
RNA extraction and reverse transcription polymerase chain reaction
RNA samples were collected from ADSCs and BMSCs after osteogenic and adipogenic differentiation at 7, 14, and 21 d of culture. RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's protocol. The RNA was resuspended in DEPC-H2O, and the RNA concentration was calculated by a spectrophotometer (NanoDrop 1000; Thermo Fisher Scientific). The method of preparing the cDNA was adjusted according to the recommendations of the MMLV Reverse Transcription Kit (Protech Technology Enterprise, Taiwan).
mRNA expression was assessed by the reverse transcription polymerase chain reaction (PCR). The primer pairs were Runx2 (forward: 5'-CCGCACGACAACCGCACCAT-3'; reverse: 5'-CGCTCCGGCCCACAAATCTC-3'), Opn (forward: 5'-ATGAGATTGGCAGTGATT-3'; reverse: 5'-GTTGACCTCAGAAGATGA-3'), and Gapdh (forward: 5'-GAGGGGCCATCCACAGTCTT-3'; reverse: 5'-TTCATTGACCTCAACTACAT-3'). After 30 cycles of PCR, the PCR product was converted from the gel image into surface area by Quantity One 1-D analysis software (Bio–Rad Laboratories, USA).
Colony-forming unit fibroblast analysis of ADSCs and bone marrow stem cells
The ADSCs and BMSCs were plated at 1 × 104 cells in a 10-cm dish and cultured until 7 d with 2 replacements of fresh medium. After differential culture, the cells were washed with DPBS, and then 0.5% crystal violet (Sigma–Aldrich) was added to stain for 5 min. The stain solution was removed, the cells were washed with DPBS, and the dish was placed in a fume hood to air dry. The number of colonies formed was analyzed by Infinity software (Vilber Lourmat, Germany). Visible colonies with a diameter >2 mm were counted. Each set of experiments had at least 3 replicates.
Comparison of the cell growth rate, colony-forming unit fibroblast formation, and osteogenesis of ADSCs and bone marrow stem cells
ADSCs and BMSCs at passage 1 (P1) and passage 3 (P3) were purified and used to determine the cell growth rate. A total of 1 × 105 cells were collected and spread in a 6-well culture plate and then cultured for 3 d and 5 d to count the cell number and calculate the cell growth rate. The cell doubling time was calculated by converting the number of cells and time through the formula:
Cell doubling time (h) = (T − T0) × log 2/(log N/N0)
Where T0 and N0 are the initial time and cell number, respectively, and T and N are the current time and cell number, respectively.
The P1 and P3 generations of the stem cells purified from ADSCs and BMSCs were tested for CFU-F.
ADSCs and BMSCs were plated on 6-well plates at 1 × 105 cells/well. When the cell density reached 80%, they were replaced with osteogenic differentiation medium for the induction of osteogenesis. The cells were harvested on 14 d for alizarin red S staining.
| Results|| |
The morphology and characteristics of ADSCs and bone marrow stem cells
ADSCs and BMSCs were harvested and differentiated from the GFP transgenic mouse, FVB/NCrl-Tg(Pgk1-EGFP)01Narl. From the embryo to the adult stage, this transgenic mouse stably expresses GFP in all tissues. After in vitro culture and purification, the small rounded and flattened cells disappeared gradually after subsequent passages. Fibroblast-like and spindle-shaped cells became predominant. Under an inverted microscope, ADSCs were observed to be similar in shape to BMSCs, but ADSCs were slightly larger, longer and flat compared to BMSCs [Figure 1]a. EGFP was stably expressed in both ADSCs and BMSCs. These stem cells expressing EGFP made subsequent experiments more convenient for observation [Figure 1]a.
|Figure 1: Morphology and characteristics of ADSCs and BMSCs. Microscopy observations of ADSCs and BMSCs collected from FVB/NCrl-Tg(Pgk1-EGFP)01Narl mice. (a) ADSCs and BMSCs under bright-field and FITC fluorescent fields. The cell surface antigens of (b) ADSCs and (c) BMSCs were immunostained with mesenchymal stem cell-positive antibodies against CD44, CD90, CD105, and Sca-1, and the images were observed under confocal fluorescence microscopy.|
Click here to view
A test was performed using immunofluorescence staining to detect the positive surface antigens of MSCs. The detection of the surface antigens on the cell membrane confirmed that ADSCs and BMSCs retained the characteristics of MSCs. Excitation wavelengths of 400 nm and 488 nm were used and collected with the specific emission wavelengths of EGFP (500 nm ~ 550 nm), PE (560 nm ~ 600 nm) and PE/Cy5 (650~700 nm) in the laser scanning confocal microscopy.
Both isolated ADSCs and BMSCs not only exhibited antibody fluorescence of CD44, CD90, CD105 and Sca-1 (antigens specific to MSCs) but also expressed autologous green fluorescence of the GFP from the transgenic mouse FVB/NCrl-Tg(Pgk1-EGFP)01Narl. The results indicate that ADSCs and BMSCs with EGFP expression retained the characteristics of MSCs cultured in vitro [Figure 1]b and [Figure 1]c.
Comparison of cell growth rate between ADSCs and bone marrow stem cells
A high growth rate is one of the characteristics of MSCs, which is helpful for in vitro culture, expansion and subsequent applications, such as stem cell therapy. This experiment intended to compare the growth rate of ADSCs and BMSCs. The experiments were performed using P1 and P3 ADSCs to compare growth rates with P1 and P3 BMSCs.
The cell doubling time of ADSCs at P1 was approximately 46 h; at P3, it was 54 h. The cell doubling time of BMSCs was approximately 84 h (P1) and 134 h (P3). The results show that the cell growth rates of P1 and P3 ADSCs were significantly higher than those of the P1 and P3 BMSCs after 3 d and 5 d of culture (P < 0.001). On day 5, the cell growth of P1 ADSCs was significantly higher than that of P3 ADSCs (P < 0.01); a similar trend was also observed in BMSCs, i.e., the cell growth of P1 BMSCs was significantly higher than that of P3 BMSCs (P < 0.05) [Figure 2]a. In addition, the growth rate of ADSCs was higher than that of BMSCs (P < 0.001), and the growth efficiency of P1 was higher than that of P3 for both ADSCs and BMSCs.
|Figure 2: Proliferation rate and colony-forming unit fibroblast analysis of ADSCs and BMSCs. Comparison in the (a) cell proliferation rate and (b) colony-forming unit fibroblast experiment to analyze ADSCs and BMSCs for mesenchymal stem cell characteristics in vitro. At least, three repeats were performed for each group in the experiment; * P < 0.05, ** P < 0.01, *** P < 0.001.|
Click here to view
Comparison of colony-forming unit fibroblast analysis between ADSCs and bone marrow stem cells
In low-density MSC culture, discrete colonies of adherent fibroblast-like MSCs are formed, and the CFU-F represents a colony that arises from a single precursor cell. More colonies formed in the CFU-F test indicate that there are more stem cells in the cell population and that their purity is higher.
The experimental results showed that the number of CFU-F colonies formed in P1 and P3 ADSCs was 23.0 ± 4.6 and 27.3 ± 5.5, and that of P1 and P3 BMSCs was 10.3 ± 1.2 and 15.0 ± 3.0, respectively [Figure 2]b. For both ADSCs (P = 0.354) and BMSCs (P = 0.066), the results showed that CFU-F tended to increase as the number of generations increased from P1 to P3. The number of CFU-F of ADSCs was higher than that of BMSCs, and the purity of ADSCs was also higher than that of BMSCs. The increase in CFU-F numbers of P3 ADSCs and BMSCs was slightly higher than that of P1 ADSCs and BMSCs, indicating that the cell purity of ADSCs and BMSCs may be improved by in vitro passage culture.
Analysis of stem cell differentiation ability
Stem cells have the characteristics of differentiating into many types of cells. Therefore, the analysis of stem cell differentiation ability is one of the ways to evaluate the quality and potential of stem cells. We used ADSCs and BMSCs to induce differentiation into adipose cells and osteoblasts. ADSCs [[Figure 3]a-1] and BMSCs [[Figure 3]a-4] were cultured and induced in adipocyte differentiation medium for 7 d. During the process of differentiation of stem cells into adipocytes, fatty acids were produced, which accumulated in the cells in large quantities and produced oil droplets present in ADSCs [[Figure 3]a-2] and BMSCs [[Figure 3]a-5]. Further experimental results of oil Red O staining showed that both ADSCs [[Figure 3]a-3] and BMSCs [[Figure 3]a-6] could successfully differentiate into adipocytes under appropriate stimulation.
|Figure 3: Adipogenic and osteogenic differentiation of ADSCs and BMSCs. (a) ADSCs and BMSCs were cultured until 80% confluence, changed to adipogenesis medium and cultured for 7 d. (1) ADSCs cultured in normal medium; (2) ADSCs induced to differentiate into adipocytes, and (3) ADSCs induced to differentiate into adipocytes, as assessed by Oil red O staining. (4) BMSCs cultured in normal medium; (5) BMSCs induced to differentiate into adipocytes, and (6) BMSCs induced to differentiate into adipocytes, as assess by Oil red O staining. Black arrows indicate adipocytes. (b) ADSCs and BMSCs were cultured until 80% confluence, changed to osteogenesis medium and cultured for 14 d. (7) ADSCs cultured in normal medium; (8) ADSCs induced to differentiate into osteoblasts, and (9) ADSCs induced to differentiate into osteoblasts, as assessed by alizarin red S staining. (10) BMSCs cultured in normal medium; (11) BMSCs induced to differentiate into osteoblasts, and (12) BMSCs induced to differentiate into osteoblasts, as assessed by alizarin red S staining. Yellow arrows indicate the osteoblasts.|
Click here to view
ADSCs [[Figure 3]b-7] and BMSCs [[Figure 3]b-10] were cultured in osteogenic differentiation medium for 7 d to induce differentiation. During the process of differentiation of stem cells into osteoblasts, calcium ions in the culture environment were precipitated with calcium phosphate. The minerals eventually accumulate on the extracellular matrix, and cell mineralization causes the crystals to appear on and around the cell surface. Mineralized ADSCs [[Figure 3]b-8] and BMSCs [[Figure 3]b-11] were observed in the osteogenic differentiation culture. Further experimental results of alizarin red S staining showed the degree of differentiation of stem cells into osteoblasts after 14 d of osteogenic differentiation, and both ADSCs [[Figure 3]b-9] and BMSCs [[Figure 3]b-12] were successfully differentiated into osteoblasts. Because cetylpyridinium chloride solution can dissolve alizarin red S dye, the concentration of alizarin red S can be measured by a spectrophotometer to indirectly determine the degree of cell mineralization, which can be used to estimate cell mineralization. The ADSCs and BMSCs were subjected to a 14 d osteogenic differentiation test, stained with alizarin red S, and then subjected to a mineralization test. The results showed that the absorption value at OD570 of ADSCs (0.72 ± 0.04) was higher than that of BMSCs (0.58 ± 0.01) (P < 0.05). The mineralization of ADSCs was better than that of BMSCs [Figure 4].
|Figure 4: Mineralization analysis of osteogenesis in ADSCs and BMSCs. Analysis of ADSC and BMSCs mineralization ability in osteogenesis medium. ADSCs and BMSCs were cultured in osteogenesis medium for 14 d, followed by alizarin red S staining, and then CPC was used to measure the OD570. At least three repeats were performed for each group in the experiment; * P < 0.05.|
Click here to view
Analysis of osteogenic differentiation-related gene expression
When osteogenesis is induced, the gene expression level and the differentiation stage of stem cells can be detected by the expression of genes related to osteogenic differentiation. After the ADSCs and BMSCs differentiated into osteoblasts, cellular RNA was extracted at 0, 7, 14, and 21 d and used to assess Runx2 and Opn gene expression by reverse transcription PCR. The Runx2 gene is a specific gene expressed in the early stage, and the Opn gene is expressed in the late stage of differentiation. Both were assessed to confirm the osteogenic differentiation stage of ADSCs and BMSCs.
The results showed that during the osteogenic differentiation process of ADSCs, Opn gene expression gradually increased from 0 d until 21 d of differentiation, and Runx2 gene expression peaked at 14 d and then rapidly decreased until 21 d, indicating that osteogenesis was near the completion stage [Figure 5]. During the differentiation process of BMSCs, the pattern of Opn gene expression was similar to that of ADSCs, but it reached a peak rapidly on Day 7 and maintained a high level until Day 21. Runx2 expression in BMSCs showed a downward trend from Day 0 until Day 21. These results indicate a similar progression of osteogenic differentiation of ADSCs and BMSCs.
|Figure 5: Osteogenesis expression of ADSCs and BMSCs. Expression of osteogenesis genes (a) Runx2 and (b) Opn analyzed in ADSCs and BMSCs at different times. ADSCs and BMSCs cultured in osteogenesis medium. Reverse transcription polymerase chain reaction was used to test gene expression at 0, 7, 14 and 21 d. The gene/Gapdh ratio represented the relative expression of genes. At least 4 repeats were performed for each group in the experiment.|
Click here to view
| Discussion|| |
Regenerative medicine is an emerging medicine for repairing aging and damaged tissues. Stem cells are stimulated to produce new tissues to treat damaged parts. Therefore, the development of stem cell therapy is one of the important methods in regenerative medicine. MSCs exist in a variety of tissues. Due to the differences in the growth of tissues, MSCs in different tissues retain their unique cell growth characteristics. This study explored the difference in characteristics between ADSCs and BMSCs.
Comparing the collection methods of different stem cells, BMSCs are derived from the bone marrow cavity and must be collected through the bone, while ADSCs are derived from adipose tissue and are easier to collect with minor surgery. Although the number of stem cells is much lower than that of mature and differentiated cells, they can be found in various tissues and organs in the body. Comparing the differences in the growth environment of stem cells in the body, the bone marrow cavity contains hematopoietic cells in addition to BMSCs, but in adipose tissue, most of the cells that exist are adipocytes. Hematopoietic stem cells are present in the culture medium as a suspension. However, when mixed with bone marrow cells, the adhesion factors secreted by BMSCs cause hematopoietic stem cells and BMSCs to attach to the plate and grow together, resulting in a decrease in the purity of BMSCs. Therefore, the collection of BMSCs needed to undergo specific cell purification processing to remove most of the hematopoietic cells to increase the content of BMSCs. The easy and efficient method we used was modified from a previous study., After removing nonadherent cells in the first 3 h, the medium was replaced with 1.5 mL fresh medium every 8 h for up to 72 h. The BMSC purity and quality increased and were suitable for the following studies.
The source of ADSCs and BMSCs in this experiment came from the transgenic mouse FVB/NCrl-Tg(Pgk1-EGFP)01Narl, which EGFP throughout the entire body, including in the stem cells. This feature was beneficial for observation during this experiment [Figure 1]. After passaging 2–3 times, both ADSCs and BMSCs displayed a more homogenous fibroblast-like, adherent, elongated, and spindle-shaped morphology cell type, which has been referred to in many reports.
To ensure that the ADSCs and BMSCs used in the experiment retained the characteristics of MSCs, we performed immunofluorescence staining on the stem cells to identify the surface antigens with specific stem cell characteristics. The literature notes that mouse MSCs specifically express the antigens CD44, CD90, CD105, and Sca-1. CD44 is a cell adhesion factor that is mainly localized in the cytoplasm and at the edge of the cell membrane, while CD90, CD105 and Sca-1 are found throughout the whole cell. In this experiment, the antibodies CD44, CD90, CD105, and Sca-1 were selected to distinguish them from transgenic mouse stem cells that expressed green fluorescence. As observed by a confocal microscope, it was found that the stem cells appeared as a yellow–green fluorescence mixture after binding these specific antibodies. Our results show that both ADSCs and BMSCs in this experiment could express MSC-specific antigens when cultured in vitro, which proved that the characteristics of MSCs were still maintained during the test. GFP stem cells also continued to be stably expressed during the experiment. In subsequent experiments, the characteristic of the green fluorescence was used to identify the growth position and performance of the stem cells.
The stem cell growth rate, differentiation potential, immune rejection, and other stem cell characteristics all affect the effectiveness of using stem cells for therapeutic applications., In the growth rate comparison experiment in the present study, the number of stem cells was calculated at different time points, and the calculation results were drawn into a growth curve diagram [Figure 2]a. The results clearly show that the growth rate of ADSCs was higher than that of BMSCs. The doubling time of ADSCs was only half the time of BMSCs [Figure 2]a. A similar result showing that ADSCs have a higher proliferation rate and longer culture survival period than BMSCs has been reported. In addition, it has been reported that the proportion of stem cells recovered from bone marrow is only 0.001 ~ 0.01%, but in stem cells recovered from adipose tissue, the recovery rate can reach 10%. We believe that in the application of stem cell therapy, a higher recovery rate and growth rate could help cell proliferation and achieve better therapeutic effects.
The CFU-F test is used for MSCs that aggregate to form colonies when cultured at a low density. When there are more stem cells present in the cell population, the colonies of cell aggregation are more obvious. Therefore, the number of colonies represents the purity of the stem cells. Our results show that the number of P3 CFU-F in BMSCs and ADSCs was higher than that of P1 CFU-F [Figure 2]b, which proved that the purity of stem cells can be improved after several generations of in vitro culture. BMSCs comes from the bone marrow cavity, which is rich in a large number of hematopoietic cells, while adipose tissue had fewer blood vessels to pass through, and the content of hematopoietic cells is lower. In the literature, it has been pointed out that during the primary culture of MSCs, too many hematopoietic cells will reduce the growth space of MSCs. This is likely the reason why the number of CFU-F in P1 and P3 of ADSCs was higher than that of BMSCs [Figure 2]b. The purity of stem cells was related to the accuracy of the test and even affected the subsequent repair effect.
We used alizarin red S staining to mark the crystals of cell mineralization. The alizarin red S dye was redissolved in cetylpyridinium chloride solution, and the absorbance value was measured with OD570 to determine the degree of stem cell mineralization. Our results show that the absorbance value of ADSCs was significantly higher than that of BMSCs [Figure 4], indicating that ADSCs produced more mineralized crystals during the process of osteogenic differentiation and that the mineralization effect of ADSCs was better than that of BMSCs.
We used Runx2 and Opn gene expression and mineralization tests to further explore and analyze the osteogenic differentiation ability of ADSCs and BMSCs [Figure 5]. Runx2 was expressed in the early stage of osteogenic differentiation and was responsible for turning on downstream genes of osteogenic differentiation, and the expression of Runx2 decreased over time (~21 d)., Opn appeared in the middle stage of osteogenic differentiation and continued to be expressed in the later stage. The expression of Opn was related to calcium ion precipitation during the mineralization process. In the final stage of osteogenic differentiation, cells are mineralized and undergo programmed death during the process of cell mineralization, and gene expression declines when entering cell mineralization. Our experiment shows similar results to other studies, in which BMSCs showed a decline in the expression of the Runx2 gene from the beginning, and ADSCs showed a decline starting at 14 d, indicating that the stem cells have entered the preosteoblast period of differentiation. Both ADSCs and BMSCs expressed Opn in osteogenic differentiation culture, which continued to increase expressed till the later stage, indicating that stem cells entered the anaphase of osteogenic differentiation. The difference was that BMSCs reached the peak of Opn expression quickly at day 7 compared to ADSCs, which lasted until day 21.
| Conclusion|| |
MSCs are multipotent stem cells that have gained significant attention in the field of regenerative medicine. ADSCs and BMSCs were purified from the transgenic mouse FVB/NCrl-Tg(Pgk1-EGFP)01Narl, which expresses the green fluorescent protein gene, were shown to carry the positive markers CD44, CD90, CD105 and Sca-1, and had the same characteristics as mouse MSCs. In the pluripotent differentiation test, both ADSCs and BMSCs were induced to differentiate into adipocytes and osteocytes. It was proven that both ADSCs and BMSCs have the potential to differentiate into multiple types of cells with the appropriate stimulation. In the CFU-F test, the number of ADSC colonies was greater than that of BMSC colonies, indicating that a greater quantity and higher purity of MSCs can be obtained from the adipose tissue. Moreover, compared to the two kinds of MSCs, ADSCs have a higher growth rate than BMSCs. The results of this study suggested that ADSCs, similar to BMSCs, might be suitable for in vitro studies when mouse MSCs are used.
The authors are grateful for the support by Ministry of Science and Technology, Taiwan (MOST 103-2313-B-415-015-MY3).
Financial support and sponsorship
Ministry of Science and Technology, Taiwan (MOST 103-2313-B-415-015-MY3).
Conflicts of interest
There are no conflicts of interest
| References|| |
Kolf CM, Cho E, Tuan RS. Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: Regulation of niche, self-renewal and differentiation. Arthritis Res Ther 2007;9:204.
Mohamed-Ahmed S, Fristad I, Lie SA, Suliman S, Mustafa K, Vindenes H, et al
. Adipose-derived and bone marrow mesenchymal stem cells: A donor-matched comparison. Stem Cell Res Ther 2018;9:168.
Fu X, Liu G, Halim A, Ju Y, Luo Q, Song AG. Mesenchymal stem cell migration and tissue repair. Cells 2019;8:E784.
Keshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cell-derived extracellular vesicles: Novel frontiers in regenerative medicine. Stem Cell Res Ther 2018;9:63.
Park SJ, Kim RY, Park BW, Lee S, Choi SW, Park JH, et al
. Dual stem cell therapy synergistically improves cardiac function and vascular regeneration following myocardial infarction. Nat Commun 2019;10:3123.
Wang T, Xu Z, Jiang W, Ma A. Cell-to-cell contact induces mesenchymal stem cell to differentiate into cardiomyocyte and smooth muscle cell. Int J Cardiol 2006;109:74-81.
Tsuchiya A, Takeuchi S, Watanabe T, Yoshida T, Nojiri S, Ogawa M, et al
. Mesenchymal stem cell therapies for liver cirrhosis: MSCs as “conducting cells” for improvement of liver fibrosis and regeneration. Inflamm Regen 2019;39:18.
Hwang S, Hong HN, Kim HS, Park SR, Won YJ, Choi ST, et al
. Hepatogenic differentiation of mesenchymal stem cells in a rat model of thioacetamide-induced liver cirrhosis. Cell Biol Int 2012;36:279-88.
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al
. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143-7.
Kang BJ, Ryu HH, Park SS, Koyama Y, Kikuchi M, Woo HM, et al
. Comparing the osteogenic potential of canine mesenchymal stem cells derived from adipose tissues, bone marrow, umbilical cord blood, and Wharton's jelly for treating bone defects. J Vet Sci 2012;13:299-310.
James AW. Review of signaling pathways governing MSC osteogenic and adipogenic differentiation. Scientifica (Cairo) 2013;2013:684736.
Stein GS, Lian JB, van Wijnen AJ, Stein JL, Montecino M, Javed A, et al
. Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene 2004;23:4315-29.
Prince M, Banerjee C, Javed A, Green J, Lian JB, Stein GS, et al
. Expression and regulation of Runx2/Cbfa1 and osteoblast phenotypic markers during the growth and differentiation of human osteoblasts. J Cell Biochem 2001;80:424-40.
Harada H, Tagashira S, Fujiwara M, Ogawa S, Katsumata T, Yamaguchi A, et al
. Cbfa1 isoforms exert functional differences in osteoblast differentiation. J Biol Chem 1999;274:6972-8.
Singh M, Foster CR, Dalal S, Singh K. Osteopontin: Role in extracellular matrix deposition and myocardial remodeling post-MI. J Mol Cell Cardiol 2010;48:538-43.
Wei J, Karsenty G. An overview of the metabolic functions of osteocalcin. Rev Endocr Metab Disord 2015;16:93-8.
National Research Council (U.S.). Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (U.S.), National Academies Press (U.S.). Guide for the Care and Use of Laboratory Animals. Washington, D.C.: National Academies Press; 2011.
Soleimani M, Nadri S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat Protoc 2009;4:102-6.
Sugii S, Kida Y, Berggren WT, Evans RM. Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells. Nat Protoc 2011;6:346-58.
Eslaminejad MB, Nikmahzar A, Taghiyar L, Nadri S, Massumi M. Murine mesenchymal stem cells isolated by low density primary culture system. Dev Growth Differ 2006;48:361-70.
Sung JH, Yang HM, Park JB, Choi GS, Joh JW, Kwon CH, et al
. Isolation and characterization of mouse mesenchymal stem cells. Transplant Proc 2008;40:2649-54.
Lee JY, Kim CK, Choi D, Park BK. Volume doubling time and growth rate of renal cell carcinoma determined by helical CT: A single-institution experience. Eur Radiol 2008;18:731-7.
Wu HT, Chou CK, Hung YC, Yu CK. Allotransplantation of transgenic mouse ovaries expressing enhanced green fluorescent protein under the control of the murine phosphoglycerate kinase 1 promoter. Reprod Domest Anim 2010;45:900-6.
Kuznetsov SA, Mankani MH, Bianco P, Robey PG. Enumeration of the colony-forming units-fibroblast from mouse and human bone marrow in normal and pathological conditions. Stem Cell Res 2009;2:83-94.
Boonrungsiman S, Gentleman E, Carzaniga R, Evans ND, McComb DW, Porter AE, et al
. The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation. Proc Natl Acad Sci U S A 2012;109:14170-5.
Chen HC, Awale S, Wu CP, Lee HH, Wu HT. Co-cultured bone marrow mesenchymal stem cells repair thioacetamide-induced hepatocyte damage. Cell Biol Int 2020;44:2459-72.
Zhou LN, Wang JC, Zilundu PL, Wang YQ, Guo WP, Zhang SX, et al.
A comparison of the use of adipose-derived and bone marrow-derived stem cells for peripheral nerve regeneration in vitro
and in vivo
. Stem Cell Res Ther 2020;11:153.
Taléns-Visconti R, Bonora A, Jover R, Mirabet V, Carbonell F, Castell JV, et al
. Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells. World J Gastroenterol 2006;12:5834-45.
Liao HT, Chen CT. Osteogenic potential: Comparison between bone marrow and adipose-derived mesenchymal stem cells. World J Stem Cells 2014;6:288-95.
Hsiao FS, Cheng CC, Peng SY, Huang HY, Lian WS, Jan ML, et al
. Isolation of therapeutically functional mouse bone marrow mesenchymal stem cells within 3 h by an effective single-step plastic-adherent method. Cell Prolif 2010;43:235-48.
Liu HY, Chiou JF, Wu AT, Tsai CY, Leu JD, Ting LL, et al
. The effect of diminished osteogenic signals on reduced osteoporosis recovery in aged mice and the potential therapeutic use of adipose-derived stem cells. Biomaterials 2012;33:6105-12.
Aubin JE. Regulation of osteoblast formation and function. Rev Endocr Metab Disord 2001;2:81-94.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]