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Original article

Human adipose-derived mesenchymal stem cells: a better cell source for nervous system regeneration

Han, Chao; Zhang, Liang; Song, Lin; Liu, Yang; Zou, Wei; Piao, Hua; Liu, Jing

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doi: 10.3760/cma.j.issn.0366-6999.20120064
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Abstract

Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into a variety of cell types including osteoblasts, chondrocytes, and adipocytes.1,2 MSCs also have the ability to differentiate into nerve-like tissues such as neurons and glial cells.3 The nervous system, unlike other systems such as the musculoskeletal and hematopoietic system, seems to be incapable of self-repair after injury. MSCs are being considered for application in neural repair since they can differentiate into neurons after appropriate induction in vitro.4 Stem cell therapy is likely to be an effective means for therapeutic nervous system repair.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been anticipated for use in transplantation therapy for various neurological disorders.5 There is evidence that these cells have the ability to differentiate into neurons and glial cells in neural degenerative diseases6 and to secrete neuroprotective factors to inhibit neuron death.7 Although bone marrow was the first and has been the main source of multipotent MSCs, the harvest of bone marrow is an invasive procedure and the number, differentiation potential, and maximal life span of BMSCs declines with increasing age.8 It has been reported that MSCs can also be isolated from muscle and adipose tissue of the synovium and periosteum.9,10 Recently, adipose tissue was identified as a promising alternative source of MSCs since adipose tissue is distributed in large amounts throughout the body and is readily obtained using liposuction or lipectomym, which are much easier and less invasive than bone marrow extraction. It has been reported that ADSCs, like BMSCs, have the capability to differentiate in vitro towards osteogenic, chondrogenic, and adipogenic lineages when cultured in the appropriate conditions.11 However, whether ADSCs have the same ability as BMSCs to differentiate into neurons after transplantation into the brain still remains undetermined.

In this study, we hypothesized that MSCs from human adipose tissue may have the same potential for therapeutic applications in nervous system diseases as do bone marrow derived MSCs. Here, we isolated MSCs from human adipose tissue and bone marrow, and studied the differences with regard to cell morphology, surface markers, neuronal differentiation capacity by using conditioned medium derived from the different adult rat brain regions, and we focused on synapse structure formation and the secretion of neurotrophic factors.

METHODS

Materials and reagents

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and fetal calf serum (FCS) were purchased from Gibco (USA) and Percoll was purchased from Pharmacia (USA). Basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and epidermal growth factor (EGF) were purchased from Peprotech (USA), and trypsin and ethylene diamine tetraacetic acid (EDTA) were purchased from Amresco (USA). CD34, CD90 (fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody), CD13, CD45, CD106 (phycoerythrin (PE)-conjugated goat anti-mouse IgG antibody) and Nissl Fluorescent dye were purchased from Invitrogen and Hoechst 33258, nestin (rabbit anti-human IgG antibody), neuron-specific enolase (NSE, rabbit anti-human IgG antibody), β-III-tubulin (rabbit anti-human IgG antibody), bovine serum albumin (BSA) and goat anti-rabbit IgG-FITC secondary antibody were purchased from Sigma, USA. Sodium azide (NaN3), paraformaldehyde and poly-l-lysine were purchased from Amersco.

Cell isolation and culture

Samples of human adipose tissue (10 ml) were collected from five donors (20-45 years old) undergoing liposuction surgery with an informed written consent, under the requirements of the Hospital Ethics Committee. Liposuction tissues were washed at least three times with sterile phosphate-buffered saline (PBS), and digested with an equal volume of 0.1% collagenase Type I and 0.25% trypsin (1:1) for 30 minutes at 37°C with intermittent shaking. The floating adipocytes and liquid supernatant were separated from the stromal vascular fraction by centrifugation (1200 r/min for 5 minutes). The cellular pellet was washed in PBS twice (1000 r/min for 10 minutes) and then resuspended with H-DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, and plated in tissue culture flasks at 2×106 cells/cm2. Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C and nonadherent cells were removed 48 hours after seeding. About 5-10 ml of adult human bone marrow samples were harvested from routine surgical procedures (pelvic osteotomies; six samples; age 35-60 years) with informed written consent. The bone marrow aspirates were added to a sterile tube containing heparin and diluted with L-DMEM. The suspension was centrifuged (1000 r/min, 5 minutes) and resuspended in L-DMEM. The mononuclear cells were isolated by density gradient centrifugation at 3000 r/min for 20 minutes using Percoll (density, 1.073 g/L) and placed at 2.0×106 cells/ml in a 25 cm2 flask in L-DMEM medium containing 10% FCS, 100 U/ml penicillin and 100 g/ml streptomycin. The culture medium was replaced twice a week and non-adherent cells were removed.

The primary culture for hADSCs and hBMSCs took 7-10 and 10-15 days, respectively, until they reached confluence. When cells became almost 80%-90% confluent, the adherent cells were detached with 0.25% trypsin solution for 5 minutes at 37°C and subcultured at 1×104 cells/cm2 for future use.

Cell characterization

hADMSCs and hBMSCs were harvested into a single cell suspension at passage 5 and stained with antibodies including anti-human CD13, CD34, CD45, CD90, and CD106 (Becton Dickinson, USA). Cells were incubated with monoclonal anti-CD13-PE, anti-CD34-FITC, anti-CD45-PE, anti-CD90-FITC, and anti-CD106-PE antibodies for 30 minutes in the dark. Flow cytometric analysis was performed with a FACSAriaII flow cytometer (Becton Dickinson).

Cell proliferation assay

hADSCs and hBMSCs at passage 5, before and after cryopreservation, were seeded in 24-well culture plates at a density of 5×104 cells/well (n=5 each). Cells were collected from each well 1-7 days after seeding and counted everyday using a Cell Counter (Beckman Coulter). Population doubling (PD) was calculated as PD=log2 (number of harvested cells/number of seeded cells).

Osteogenic and adipogenic differentiation potential

For osteogenic differentiation, cells were seeded in 35 mm2 culture dishes at a density of 1×105/cm2 and cultured for four weeks with 100 nmol/L dexamethasone, 50 mol/L ascorbic acid, and 10 mmol/L β-glycerophosphate. Osteogenic differentiation was evaluated by Von Kossa staining according to Sheehan's method.12 For adipogenic differentiation, cells were cultured in the presence of 1 μmol/L dexamethasone, 0.2 mmol/L indomethacin, 10 g/ml insulin and 0.5 mmol/L 3-isobutyl-1-methylxanthine. After three weeks of culture, cells were fixed with 4% paraformaldehyde and stained with 3 mg/ml Oil Red O (Sigma) dissolved in 60% isopropanol for 10 minutes and the excess dye was washed out with water.

Rat brain-conditioned medium preparation

Rats, 3-4-months old, were anesthetized and sacrificed and the cerebellum, hippocampus, and cortex tissue was removed at 4°C and placed into 6 ml DMEM culture medium under sterile conditions. The medium was placed in a 4°C refrigerator for 2 hours and stirred, then the soluble fraction was collected (10 000 r/min, 30 minutes, 4°C) and filtered through a 0.22-μm membrane for sterilization. Protein content was detected with Coomassie brilliant blue at a concentration of 200 ng/ml and stored at -80°C.

Neuronal differentiation

Cells at passage 5 were seeded into 35 mm2 culture dishes and cell morphology was observed after 24 hours. When cells reached 60% confluence, the medium was replaced with rat brain-conditioned medium. The cells were then fixed and neuron-specific markers were detected at time points from Day 1 to Day 14 during neuronal differentiation.

For chemical induction, hMSCs were preincubated for 24 hours with Stage I media, consisting of H-DMEM supplemented with 1% FCS, 10 ng/ml bFGF and 10 ng/ml epidermal growth factor (EGF). The cells were then incubated for 4-30 days with stage II media, composed of H-DMEM supplemented with 20 ng/ml bFGF, 20 ng/ml EGF, 20 ng/ml ciliary neurotrophic factor (CNTF) and 6 mg/ml all-trans-retinoic acid (RA) (all from Sigma, USA). hADSCs cultured in basic medium were used as control groups.

Immunofluorescence staining

Differentiated hADSCs and hBMSCs cultured on slides were fixed in 4% paraformaldehyde overnight. Cells were then incubated with primary antibodies (PBS + 3% BSA + 0.02% NaN3 + 0.2% TritonX-100) for 90 minutes at room temperature and with the secondary antibody for 60 minutes, followed by three rinses with PBS. Cell nuclei were labeled with Hoechst 33258 for 5 minutes at room temperature. The dilution rates for the antibodies used in the experiments are: NSE (rabbit anti-human 1:100), β-III-tubulin (rabbit anti-human 1:200), FITC-conjugated secondary antibody (1:70), Nissl staining (1:100), synaptophysin (1:200) and GAP-43 (1:1000). Slides were visualized by a fluorescence microscope with a digital camera (Leica, Germany) on Days 7 and 14 after neuronal differentiation. At least 10 images were randomly chosen from each staining, and the number of positive cells was counted and analyzed.

Measurement of secreted BDNF and NGF

A measured amount of (5×105) cells were seeded in 6-well plates and the induced culture medium was changed every two days. At Days 5, 8, 11, and 14, MSM-conditioned media (100 μl) was collected and stored at -80°C. BDNF and NGF secretions were tested with enzyme-linked immunosorbent assay (ELISA) kits (Bethyl Laboratories, TX, USA) according to the manufacturer's instructions. All samples were analyzed in triplicates.

Statistical analysis

Data are presented as mean ± standard deviation. Results were analyzed by the Student's t-test. SPSS13.0 software (SPSS Inc., IL, USA) was used for data analysis with P ≤0.05 indicating significant difference.

RESULTS

Cell isolation and characterization

Primary hADSCs (Figure 1A) and hBMSCs (Figure 1B) showed characteristic fibroblastic-like morphology when attachment to plastic plates. The cell bodies of hBMSCs appeared larger than that of hADSCs. hADSCs and hBMSCs became relatively homogeneous in appearance as passages progressed. At passage five, the cells demonstrated a flat and fusiform appearance (Figure 1C and D).

Figure 1.
Figure 1.:
Morphology of hADSCs and hBMSCs. A: hADSC; B: hBMSC primary cultures demonstrated a flat and fusiform appearance. C: hADSCs; D: hBMSCs of passage 5 at near confluence.

Flow cytometry was performed using antibodies against MSC surface markers (Figure 2). The results showed that approximately 95% of hBMSCs were positive for CD13 and CD90. 50% were positive for CD106 and negative for the hematopoietic stem cell markers CD34 and CD45. hADSCs were positive for CD13, CD90, and negative for CD34, CD45 and CD106. hADSCs and hBMSCs showed a typical mesenchymal-like immunophenotype.

Figure 2.
Figure 2.:
Flow cytometric analysis of surface adhesion molecules on hADSCs and hBMSCs. Cells were labeled with monoclonal antibodies specific for molecules indicated in each flow cytometric histogram: hADSCs were positive for CD13 and CD90 but negative for CD34, CD45, and CD106. hBMSCs were positive for CD13, CD90, and CD106 but negative for CD34 and CD45.

We investigated the potential of hADSCs and hBMSCs to differentiate into osteoblasts and adipocytes using the protocols from Bertani et al.13 After cultured in osteogenic medium for four weeks and adipogenic medium for three weeks, both hADSCs and hBMSCs could differentiate into osteoblasts or adipocytes as revealed by Von Kossa or Oil Red O staining, respectively (Figure 3).

Figure 3.
Figure 3.:
Osteogenic and adipogenic differentiation of hADSCs and hBMSC. Von Kossa staining after 28 days of osteogenic induction for hADSCs (A) and hBMSCs (B). Oil Red O staining showed lipid droplets after 21 days of adipogenic induction for hADSCs (C) and hBMSCs (D). Scale bar=200 μm.

Cell proliferation

Obtaining a large number of cells in a short time is of great importance in stem cell clinical transplantation. We compared the proliferation ability of passage 5 hADSCs and hBMSCs by direct cell counting. We found that hADSCs had greater proliferation capacity than hBMSCs. The population doubling time of hBMSCs (21.9 hours) was significantly longer than that of hADSCs (16.9 hours). We also detected the growth pattern of both cell types at passage 6 after freezing and thawing. The results showed that the doubling time of hADSCs was not altered (P >0.05), while hBMSCs took significantly longer (P <0.05). The results suggested that hADSCs proliferate more quickly in vitro than hBMSCs while maintaining their proliferation capacity after cryopreservation (Figure 4).

Figure 4.
Figure 4.:
The proliferation ability of the hADSCs and hBMSCs of passage 5 and the growth curve of the recovery hADSCs and hBMSCs cells. The population doubling time of hBMSCs is 21.9 hours, hADSCs 16.9 hours. The doubling time of hADSCs was not altered (P >0.05), while hBMSCs took significantly longer (P <0.05) after freezing and thawing.

Rat brain-conditioned medium

In order to imitate the post-transplantation environment in the brain, we used rat brain conditioned medium to induce hMSCs to differentiate into neuron-like cells. Classic chemical factor induction methods were used as control. We used Nissl and NSE to confirm the effectiveness of the rat brain-conditioned medium. Morphological and immunocytochemistry results (Figure 5) showed no difference between the two induction methods (P <0.05).

Figure 5.
Figure 5.:
Phase contrast images showing morphological differences. Fluorescence images for Nissl (red) and NSE (green) are shown. Cells with multipolar morphology are present in the chemical induction medium, rat brain conditioned medium and the rat hippocampus conditioned medium.

Morphological change of differentiated cells

Morphological changes of differentiated cells were observed by phase contrast microscopy. In standard culture conditions, both confluent hADSCs and hBMSCs appeared spindle-shaped and without expression of neuronal markers (Figure 6). After seven days of differentiation with conditioned medium, both cell types assumed neuronal morphology. Fibroblastic-like cytoplasm formed a contracted multipolar cell body and left extensions peripherally. Cell bodies became increasingly pyramidal, spherical and refractile, exhibiting typical neuronal perikaryal appearance. These changes suggested that both hADSCs and hBMSCs differentiated into neuron-like cells. The number of cells that underwent morphological changes increased progressively after differentiation for 14 days.

Figure 6.
Figure 6.:
Cell morphology changed after neuronal induction as indicated by phase-contrast microscopy. Cells differentiated into neurons after 7 (A, B) and 14 (C, D) days. Cells displayed morphological features typical of neurons, such as spherical shape and extending processes. At Day 7, a portion of the cells developed spherical cell bodies with bipolar or multi-polar processes that appeared to be connected with each other. The cells displayed neuron-like shape on Day 14. Scale bar=200 μm.

Expression of neuronal markers

To investigate whether hADSCs have similar hBMSC neurogenic differentiation capabilities, we compared the expression of neuronal markers in both cell types during neuronal induction. Nestin is widely considered a specific marker of neural stem cells (NSCs) and their progenitors. Thus, nestin expression was investigated in induced MSCs as a primary marker to determine neuronal potentiality in this cell population. After seven days of neuronal induction, cells lost the characteristic morphology of MSCs and expressed high level of nestin, which then decreased after 14 days of induction. Expression of β-III-tubulin in both cell types increased from Day 0 to Day 14 after induction. The maximum percentage of cells expressing β-III-tubulin was ((61.7±1.9)%) (hADSCs) and (63.9±0.8)% (hBMSCs). After seven days of neuronal induction, cells began to express low levels of NSE; however, a significantly higher percentage of positive hBMSCs ((17.2±1.1)%) were found compared to hADSCs. After 14 days of induction, over 50% of cells were NSE positive in both groups (Figure 7). To monitor individual cell characteristics and physiological change during neuronal differentiation, Nissl stain was used with other neuronal markers in both hADSCs and hBMSCs. As Nissl bodies are peculiar to neurons, found in their cytoplasm and composed of rough endoplasmic reticulum and free polyribosomes,14 the appearance of nissl bodies is associated with mature neurons. The level of Nissl substance detected in hADSCs ((2.1±2.5)%) was much lower than in hBMSCs ((16.4±2.1)%) after seven days of differentiation. Nissl stain positive cells in both types of MSCs increased after 14 days of induction. There was no significant difference between the two cell types. From Day 7 to Day 14 after neuronal differentiation, 60%-75% of the cells exhibited typical neuronal cell morphology with axons and dendrite-like shapes. Both the expression of NSE and positive fluorescent Nissl stain were observed. The expression of NSE and formation of nissl bodies further suggested that both induced hADSCs and hBMSCs have the characteristics of neurons, although hADSCs seemed to differentiate into mature neural-like cells slower than hBMSCs.

Figure 7.
Figure 7.:
Immunocytochemistry analysis of hADSCs and hBMSCs after induction for 7 and 14 days (A-P). hADSCs and hBMSCs were stained with primary antibodies against Nestin, tubulin-III, NSE and Nissl. The percentage of positive cells was calculated and analyzed (Q, R). Scale bar = 200 μm (* P <0.05).

Synapse formation in hADSCs and hBMSCs

We investigated whether the neurons formed from hADSCs and hBMSCs in terms of synaptic structure formation. hADSCs and hBMSCs both were treated with conditioned medium for 21 days, and the cells were studied for synaptophysin (SYP) production by immunofluorescence using anti-SYP. The treated cells were also labeled for GAP-43, because it is considered a crucial component of the axon and presynaptic terminal. The studies used two groups of MSCs exposed to condition medium for 21 days, because stem cells differentiation into neuron was a long and complex process. The results showed bright fluorescence intensity for synaptophysin in two kinds of cells. In addition, GAP-43 expressed more in the site of axonal growth in hADSCs than that of hBMSCs. These results suggested that both hADSCs and hBMSCs were capable to differentiate into mature neural-like cells with synapse structure (Figure 8).

Figure 8.
Figure 8.:
Synaptophysin (A and B, arrow shown) and GAP-43 (C and D) protein expression of the induced cells. hADSCs and hBMSCs were treated with conditioned medium, and double labeled for hoechst33258 and synaptophysin/GAP-43, respectively.

Analysis of neurotrophic factor synthesis

Given previous reports suggesting that hBMSCs could secrete BDNF and NGF,15,16 we next tested whether hADSCs have the same neural secretion ability as hBMSCs during neuronal differentiation. ELISA analysis was carried out to detect the neurotrophins NGF and BDNF. Upon incubation in culture medium, an elevation in the concentration of NGF was observed in hADSCs at Day 8, followed by a decrease. Similarly, the concentration of BDNF in hBMSCs was the highest after incubation for five days and then decreased (Figure 9). The results suggested MSCs from bone marrow and adipose tissue may selectively secret and absorb different neurotrophic factors during neural differentiation.

Figure 9.
Figure 9.:
Production of BDNF and NGF by hADSCs and hBMSCs. The induced culture medium was changed every two days and then stored at -80°C. In order to detect the induced neuron secretion ability, the medium that was collected at Days 5, 8, 11, and 14 was analyzed by the BDNF and NGF ELISA kit. The control group was the induced condition culture medium (* P <0.01).

DISCUSSION

The objective of this study was to provide a comparison of human ADSCs and BMSCs as they differentiated invitro towards neuron-like cells with regards to Nissl body and synapse formation and neurotrophic factor synthesis function, and to suggest the possibility of applying hADSCs as an alternative source of stem cells for therapeutic application for neural diseases.

It has been reported that autologous hBMSC transplantation for treatment of neurological diseases has obtained some good results, but there are also some limitations for the treatment.17 Firstly, hBMSC proliferation is too slow to meet the need for transplantation. Secondly, the proliferation ability of hBMSCs decreased with the increasing age, especially for the older patients who had nervous system diseases.18,19 The limitation of the amount of bone marrow that can be obtained extended the time in culture required to generate a therapeutic cell dose.20 And it has been reported that the volume of human bone marrow taken under local anesthesia is generally limited, and cell numbers are not enough to expand to the numbers needed in the clinical usage.21 Therefore, the ideal seeding cells for stem cell therapy has become a necessary problem to solve.22

Adult adipose tissue, like bone marrow, is derived from the embryonic mesenchyme. Adipose tissue represents a rich source of MSCs, and provides an abundant and accessible source of adult stem cells with minimal patient discomfort.23 It has been found that hADSCs have the ability to widely differentiate into neural cells. Therefore, adipose tissue may be a good candidate as a source of stem cells with a therapeutic potential for neural cell therapy and regeneration. However, the detection methods of induced cells are imperfect which cannot prove the hADSCs' neural differentiation capacity completely. There is still a lack of a comprehensive comparison between hBMSCs and hADSCs differentiating into neuronal cells.24

In this study, hBMSCs and hADSCs were isolated and characterized by flow cytometry and osteogenic and adipogenic differentiation capability. MSCs can differentiate into neurons when treated with induction medium supplemented with neurotrophic factors or chemical reagents.25 Ning et al employed a neural induction medium (NIM) consisting of three active ingredients, insulin, indomethacin, and isobutylmethylxanthine (IBMX), and found that IBMX was able to induce the differentiation of ADSCs into neuron-like cells.26 Safford et al demonstrated that pretreatment with EGF and bFGF could enhance the neuronal differentiation of human ADSCs.27 However, the choice of these agents was reasonable in a limited number of studies, and there is a lack of explanation on how these compounds work together. More importantly, this induction medium could not imitate the real microenvironment when the stem cells are transplanted into the body. Li et al performed neural differentiation of ADSCs by indirect coculture with Schwann cells and found that significant neural differentiation appeared seven days later. Abouelfetouh et al studied the morphological change of BMSCs into neuron-like cells after co-culture with hippocampal slices and found that neuron-like cells tended to form network-like connections around Day 14.28 Francisco proposed that adult hippocampus-derived soluble factors induce a neuronallike phenotype in MSCs.29 To better simulate the neural microenvironment, we first used the rat brain cerebral source factors secretion-conditioned medium as the induced condition to differentiate MSCs into neural lineage in vitro. Our results suggest that after seven days of induction by rat brain-conditioned medium, the morphology and phenotype of hBMSCs changed and the cells have differentiated into neuron-like cells, and expressed neurological proteins such as nestin, β-tubulin-III and NSE, which is consistent with other reports.30 The results showed that hADSCs can also express nestin, β-tubulin-III and NSE after induction for seven days, and there is no significant difference between the positive rates of the protein expression in hADSCs and hBMSCs. In most studies, the general characteristics of induced nerve cells are identified by the expression of the immunological protein. However, the functions of these neuron-like cells are of great importance. From the view of neuron-like cell functions, the Nissl bodies, synapse formation, neurotrophic factor synthesis function and neuron-specific protein expression were studied between hADSCs and hBMSCs. Nissl bodies are dispersed in the cytoplasm of nerve cells, which can be used as specific functional indicators of the activity of nerve cells. Synaptophysin and GAP-43 are two presynaptic proteins that have long been advocated as markers of neuroplasticity.31 This study was the first study to detect Nissl body and synapsin expression in induced cells to verify the physiological function of the synaptic body. The results showed that there was more GAP-43 distributed in the site of axonal growth in induced hADSCs compared with hBMSCs, indicating there was more synapsis formation in hADSCs than in hBMSCs.

BDNF is a protein synthesized in brain, and widely distributed in the central nervous system.32 It plays an important role in neuronal survival, differentiation, growth and development. NGF has the biological functions of a dual nerve cell growth regulator and the neuronal axon growth promoting nutrition. To further identify the secretion function of induced neural-like cells, we studied the BDNF and NGF secretion in cell culture medium after differentiation using an ELISA kit. The results demonstrated the concentration of NGF in hADSC culture decreased after eight days, indicating secreted NGF had been consumed by these cells, while NGF concentration in the medium of hBMSC culture continued to increase. In addition, the concentration of BDNF in hBMSC culture increased before Day 5 and then decreased. These results revealed that there was a difference of NGF and BDNF secretion function between hADSCs and hBMSCs. Neurotrophic factors such as NGF and BDNF may play different roles in neuronal differentiation of hADSCs and hBMSCs, and these findings may provide useful knowledge for the development of neuronal differentiation methods.

Cryopreservation plays an important role in maintaining off-the-shelf availability for a variety of tissues and cells. Cryopreserved cells are likely to become the main sources for tissue engineering and stem cell therapy. If stem cells can be cryopreserved for long-term and still retain a high level of viability and potential to differentiate into tissue-specific cells, their clinical applications can be greatly simplified.33 Many researchers have demonstrated that BMSCs after cryopreservation maintained their potential for proliferation and differentiation in vitro. Shigeura et al reported that ADSCs fully retained their potential for differentiation into adipocytes, osteoblasts and chondrocytes, and for proliferation.34 Goh et al found that cryopreservation did not affect the growth and morphology of ADSCs.35 However, there is no report that the proliferation of cryopreserved human BMSCs was compared with that of ADSCs. In this study, we compared the growth curves of hBMSCs and hADSCs from three different samples before and after cryopreservation. The result showed that the hADSCs have the advantage in the proliferation ability. After cryopreservation, the recovered hADSCs still maintained the same proliferation ability as before cryopreservation, but the hBMSCs did not. These results may suggest that hADSCs are a better alternative for seeding cells with regard to cell therapy application.

There are some reports concerning the comparison of differentiation potential and the proliferation capacity of ADSCs and BMSCs. Taléns-Visconti et al differentiated human BMSCs and ADSCs into hepatogenic cells and found that ADSCs have a similar hepatogenic differentiation potential to BMSCs, but a longer culture period and higher proliferation capacity. Therefore, they pointed out that adipose tissue may be an ideal source of large amounts of autologous stem cells, and may become an alternative for hepatocyte regeneration, liver cell transplantation or preclinical drug testing. Izadpanah et al demonstrated that human MSCs from adipose tissue expanded routinely beyond 30 passages, while cells from bone marrow became senescent by passage 20.36 Comparative analysis by Golipour et al showed that the Schwann-like cell differentiation potential of ADSCs was slightly decreased in comparison with BMSCs. In the present study, we found that hADSCs have a similar neuronal differentiation potential to hBMSCs, but higher proliferation capacity before and after cryopreservation.

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Keywords:

adipose derived stem cells; bone marrow mesenchymal stem cells; neuronal differentiation; rat brain-conditioned medium

© 2014 Chinese Medical Association