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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 52  |  Issue : 5  |  Page : 17-21
 

Rabbit nucleus pulposus cells facilitate differentiation of adipose-derived stem cells into nucleus pulposus-like cells


1 Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
2 Department of Orthopedics, The Second Hospital of Tianjin Medical University, Tianjin, China

Date of Web Publication3-Nov-2015

Correspondence Address:
C W Jing
Department of Orthopedics, The Second Hospital of Tianjin Medical University, Tianjin
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.168950

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 » Abstract 

Objective: To investigate the feasibility of inducing adipose-derived stem cells (ADSCs) to nucleus pulposus cells (NPCs). Materials and Methods: ADSCs were isolated from rabbit while NPCs were isolated from an allogeneic rabbit. NPCs were co-cultured with the 3rd generation ADSCs in co-cultured system. Only NPCs were cultured in single culturing group. Through the collagen type II collagen immunohistochemistry, we observed NPCs and then identify NPC. Proteoglycan messenger RNA (mRNA) and collagen type II mRNA level were measured by real-time polymerase chain reaction. Results: In two group cells, collagen type II collagen were detected by immunohistochemistry. The amount of proteoglycan mRNA and collagen type II mRNA was both significantly higher in co-cultured group than in single cultured group. Conclusions: In some condition, ADSCs have the potency to differentiate toward nucleus pulposus-like cells. ADSCs are better seed cells for tissue engineering of artificial nucleus pulposus.


Keywords: Adipose tissue, cell culture, nucleus pulposus cells, stem cells, tissue engineering


How to cite this article:
Xu J, Qi D L, Pang X J, Jing C W. Rabbit nucleus pulposus cells facilitate differentiation of adipose-derived stem cells into nucleus pulposus-like cells. Indian J Cancer 2015;52, Suppl S1:17-21

How to cite this URL:
Xu J, Qi D L, Pang X J, Jing C W. Rabbit nucleus pulposus cells facilitate differentiation of adipose-derived stem cells into nucleus pulposus-like cells. Indian J Cancer [serial online] 2015 [cited 2021 Jul 31];52, Suppl S1:17-21. Available from: https://www.indianjcancer.com/text.asp?2015/52/5/17/168950



 » Introduction Top


Disc degenerative disease (DDD) is a common and frequently occurring illness. At present, cellular transplantation in DDD is becoming a new therapeutic method, which may reach some functional recovery through cells reconstruction. Many studies have been on bone marrow mesenchymal stem cells (BMMSCs),[1],[2] but the adipose-derived stem cells (ADSCs) treatment for DDD is not fully understood. Compared to BMMSCs, ADSCs have some advantages such as multi-differentiated potential; the sources of ADSCs are abundant; the proliferation is faster; and the time for culture growth is shorter. The study aims at exploring the ability of ADSCs to differentiate toward nucleus pulposus-like cells.


 » Materials and Methods Top


Materials

Clean, healthy New Zealand rabbits, one was 6-month-old, female, 2.5 kg; the other one was 3-month-old, female, 2.1 kg (provided by Tianjin Experimental Animal Company, Tianjin, China). Main instruments used were constant temperature CO2 incubator (Forma Scientific, USA), inverted microscope (Olympus, Japan), and desk centrifuge machine (Sigma, USA). Reagents used were Dulbecco's modified eagle medium (DMEM) (Hycolone, USA), fetal calf serum (Hycolone, USA), collagenase type I (Hycolone, USA), trypsin (Hycolone, USA), mouse anti-human CD44 and CD49d antibody (Biolegend, USA), DMEM/F12 (Hycolone, USA), and collagenase type II (Hycolone, USA).

Isolated culture of adipose-derived stem cells

The 6-month-old rabbit was anesthetized by 3% butaylone 1 ml/kg (i.m.); after anesthesia, the rabbit was fixed on operation table in prone position and sterilized. Posterior midline incision of the neck was made; about 5 ml of subcutaneous fat was cut out and put in the phosphate buffered saline (PBS) buffer solution. After soaking in PBS buffer and washing, cut into pieces of about 1 mm 3 and moved to 50 ml conical flask. Then, added 5 times of the fat volume of 0.1% collagenase type I, following a 30-min digestion at 37°C, added the same volume DMEM to stop digestion. The suspension was centrifuged at 1500 r/min for 10 min at 40°C. The cells were incubated for 36–48 h in a humidified 5% CO2 incubator until they reached approximately 80% confluency. The cells were passed at a plating proportion of 1:2 and expanded to three passages. The cells were observed by observing morphology [Figure 1] and immunofluorescence staining [Figure 2] and [Figure 3].
Figure 1: Adipose-derived stem cells of original generation, the cells are long spindle (inverted microscope, ×100)

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Figure 2: Cell membrane is green fluorescent, with positive express result (CD44 immunofluorescence stain, ×400)

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Figure 3: Cell membrane was green fluorescent, with positive express result (CD49d immunofluorescence stain, ×400)

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Isolating culture of nucleus pulposus cells

The 3-month-old rabbit was anesthetized by 3% butaylone 1 mL/kg (i.m.) and fixed on operation table in the prone position. Under sterile circumstances, the thoracolumbar spinal column was taken out, washed twice by D-HANK liquid. The vertebrae were then separated from the column one by one to get nucleus pulposus tissue. After being washed 3 times by DMEM/F12 (including 15%fetal calf serum, antiascorbic acid 30 μg/mL, penicilin 1 × 105 U/L, streptomycin 0.1 g/L, pH 7.0), nucleus pulposus was digested with agitation for 15 min in 0.25% collagenase type II at 37°C. The suspension was centrifuged at 1000 r/min for 8 min at room temperature. The cells were incubated in DMEM/F12 (including 15% fetal calf serum, antiascorbic acid 30 μg/mL, penicillin 1 × 105 U/L, streptomycin 0.1 g/L, pH 7.0) in a humidified 5% CO2 incubator. Adjusted nucleus pulposus cells (NPCs) to cells density 1 × 104/L, the 3rd generation ADSCs to 1 × 106/L. Co-culturing group: NPCs and ADSCs were co-cultivated by volume ratio 1:1. Single culturing group: Only NPCs. Both in DMEM/F12 (including 15% fetal calf serum, antiascorbic acid 30 μg/mL, penicilin 1 × 105 U/L, streptomycin 0.1 g/L, pH 7.0) in a humidified 5% CO2 incubator.

Detection of proteoglycan messenger RNA and collagen type II messenger RNA

The expression level of proteoglycan and collagen type II messenger RNA (mRNA) were detected by real-time polymerase chain reaction (RT-PCR) at times 3, 7, 14, 21 days. The primary series are given below [Table 1].[3] The RT-PCR conditions for proteoglycan: 94°C, 2 min, 94°C, 30 s, 49°C, 30 s, 72°C, 2 min, 30 cycles; the RT-PCR condition for collagen type II: 94°C, 2 min, 94°C, 30 s, 58°C, 30 s, 72°C, 2 min, 30 cycles; β-actin PCR condition: 94°C, 2 min, 94°C, 30 s, 56°C, 30 s, 72°C, 2 min, 30 cycles. Productions of PCR were detected by 1% agarose gel electrophoresis, and the net indexes were measured by Scnimage soft, which were compared with indexes of β-actin for ratios.
Table 1: Primers used for RT-PCR analysis

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Statistical analysis

These measurement data were denoted with mean ± standard deviation ( ± s), and analyzed using the SPSS 13.0 software (SPSS Inc., Chicago, IL, USA), which were tested by t-test to compare the two population mean. A P < 0.05 was considered as statistically significant.


 » Results Top


Observation by inverted microscope

Primary NPCs can be seen as round or oval-shaped [Figure 4]. The two group cells were grown in good condition. After 7 days, NPCs in co-culturing system became polygon or short fusiform shape; in the same time, ADSCs in co-culturing group has changed to polygon or short fusiform shape, in which nucleus could be seen clearly. After 14 days, cells in the two groups both became conjugated; however, cells conjugation in co-culturing group was more than single culturing group. Co-culturing cells coalesced to clump, the morphology of ADSCs was similar to NPCs and could not be distinguished [Figure 5],[Figure 6],[Figure 7],[Figure 8], so ADSCs had differentiated to NPCs-like.
Figure 4: The original generation nucleus pulposus cells primary nucleus pulposus cells were round or oval-shaped (inverted microscope, ×200)

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Figure 5: On the 14th day, co-cultured cells more coalesced to clump, could not tell adipose-derived stem cells or nucleus pulposus cells (inverted microscope, ×100)

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Figure 6: On the 14th day, single cultured nucleus pulposus cells less coalesced to clump (inverted microscope, ×100)

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Figure 7: On the 14th day of co-cultured cells, more coalesced to clump, with blue nuclei, purple in cytoplasm, could not tell adipose-derived stem cells and nucleus pulposus cells (toluidine blue stain, ×100)

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Figure 8: On the 14th day of single cultured nucleus pulposus cells less coalesced to clump, the nuclei are blue, purple in cytoplasm (toluidine blue stain, ×100)

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Toluidine blue stain

After 14 days about co-cultured group cells, more coalesced to clump, the nuclei are blue, purple in the cytoplasm, could not tell ADSCs and NPCs by toluidine blue stain [Figure 7] and [Figure 8].

Collagen type II immunohistochemistry

After 14 days about co-culturing group, cells are positive through collagen type II immunohistochemistry, with performance characteristics of NPCs, abundant cytoplasm, with visible granular material. Positive cells of collagen type II immunohistochemistry in co-culturing group significantly more than single culturing group [Figure 9] and [Figure 10].
Figure 9: On the 14th day, co-cultured cells were positive through collagen type II immunohistochemistry, with performance characteristics of nucleus pulposus cells, abundant cytoplasm, visible granular material (collagen type II immunohistochem, ×400)

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Figure 10: On the 14th day, single cultured nucleus pulposus cells were positive through collagen type II immunohistochemistry, but cell quality is less (collagen type II immunohistochem, ×400)

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Detection by real-time polymerase chain reaction

The proteoglycan mRNA and Collagen type II mRNA expression in the two groups were detected by RT-PCR on the days 3, 7, 14, and 21. In different times, the quantities of proteoglycan mRNA and Collagen II mRNA are higher in co-culturing group than in single culturing group. The contrast of the two groups total proteoglycan mRNA: t = 3.436, P < 0.05; the contrast of the two groups total Collagen II mRNA: t = 2.733, P < 0.05 [Table 2] and [Table 3], [Figure 11],[Figure 12],[Figure 13],[Figure 14].
Table 2: The quantity of expressed proteoglycan mRNA (±s)

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Table 3: The quantity of expressed collagen II mRNA (±s)

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Figure 11: The comparison about the quantity of expressed proteoglycan messenger RNA

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Figure 12: The comparison about the quantity of expressed collagen type II messenger RNA

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Figure 13: The proteoglycan messenger RNA and collagen type II messenger RNA expression in co-cultured group were detected by real-time polymerase chain reaction

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Figure 14: The proteoglycan messenger RNA and collagen type II messenger RNA expression in single cultured group were detected by real-time polymerase chain reaction

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 » Discussion Top


On suitable condition, bone marrow stromal cells (BMSCs) have some potency to differentiate toward skeletogenous cells, cartilage cells, adipose cells, etc.[4],[5] Transplantation with allogenic BMSCs cannot stimulate lymphocyte immune reaction.[4] Scientists acquired ADSCs from fat tissue, which had been differentiated toward other tissue successfully, as for bone, cartilage, skeletal muscle, fat, nerve, inducing by different cytokines.[6],[7] More study verified that ADSCs have the similar ability of multi-directional differentiation as BMSCs.[8],[9],[10]

ADSCs are abundant in tissue source compared with BMSCs.In vitro ADSCs proliferate faster, and the period is shorter than BMSCs. The ability of ADSCs for multi-directional differentiation is similar to BMSCs. ADSCs can secrete many angiogenesis factors, anti-apoptosis factors, vascular endothelial growth factor, which can influence host tissue, and ADSCs have the characters to be a lack of immunogenicity.[11] So, ADSCs can be used as seed cells for tissue engineering.

In this study, through inverted microscope, the original generation ADSCs was long spindle, and the original generation NPCs was round or oval-shaped. In the experiment, during co-culturing, the cell appearance is changing gradually. On the 7th day, ADSCs were polygon or short fusiform shape, without fiber-like changing, and NPCs became polygon or short fusiform shape too. On the 14th day, through inverted microscope, all these co-cultured cells were more coalesced to clump contrast to single cultured NPCs, and co-cultured ADSCs could not be discriminated from NPCs. On the 14th day of co-culturing group cells, more coalesced to clump, the nuclei are blue, purple in the cytoplasm, could not discriminate ADSCs and NPCs by toluidine blue stain. About co-culturing group, cells are positive through collagen type II immunohistochemistry, with performance characteristics of NPCs. Through the morphological observation, ADSCs differentiate toward nucleus pulposus-like cells gradually under the action of NPCs, in accordance with stem cell differentiation characteristics.

NPCs can produce extracellular matrix and maintain extracellular matrix in steady condition. Proteoglycan and collagen type II are phenotypes of normal NPCs. Moreover, the quantities of proteoglycan and collagen type II in nucleus decrease as soon as the discs degenerate. The quantities of proteoglycan mRNA and collagen type II mRNA expressed in co-cultured group are higher than expressed in single cultured group. Hence during co-culturing, NPCs grow better, secreting more proteoglycan and collagen type II in co-cultured group than in single cultured group. At the same time, nucleus pulposus-like cells increase the secretion of proteoglycan and collagen type II for ADSCs.[12]

During co-culturing, NPCs provided the cells environment for ADSCs differentiation, which helped ADSCs differentiate toward nucleus pulposus-like cells. ADSCs have the ability to secrete a variety of growth factors with functions similar to that of BMMSCs, so NPCs maintained good growth and proliferation condition with more secretion of proteoglycan and Collagen type II.

The experiment confirmed ADSCs nutrition effect of NPCs; ADSCs can be used to repair the nucleus pulposus degeneration. Because of ADSCs with restraining alloimmunization role,[13] after foreign heterogeneous nucleus pulposus transplanting, it is no immune rejection happened.[14],[15] ADSCs have a promising future in the study about tissue engineering nucleus pulposus as seed cells toward nucleus pulposus-like cells. However, the induction of micro-environment of NPCs in this study, how ADSCs differentiate toward nucleus pulposus-like cells is just through judgment of the appearance of cells morphology and secretion of proteoglycan and collagen type II. Hence, further research is required to exactly know its difference toward NPCs, and how ADSCs repair degenerated nucleus pulposus.


 » Acknowledgments Top


This study was supported by Tianjin Medical University Research Foundation in Tianjin Medical University, China.

 
 » References Top

1.
Chan SC, Gantenbein-Ritter B, Leung VY, Chan D, Cheung KM, Ito K. Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: A feasibility study using organ culture. Spine J 2010;10:486-96.  Back to cited text no. 1
    
2.
Allon AA, Aurouer N, Yoo BB, Liebenberg EC, Buser Z, Lotz JC. Structured coculture of stem cells and disc cells prevent disc degeneration in a rat model. Spine J 2010;10:1089-97.  Back to cited text no. 2
    
3.
Liu Z, Jia C, Han C. Experimental study on chondrogenic differentiation of rabbit adipose-derived stem cells treated with growth differentiation factor 5. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2009;23:483-9.  Back to cited text no. 3
    
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Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001;98:2396-402.  Back to cited text no. 4
    
5.
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.  Back to cited text no. 5
    
6.
Gimble I, Guilak F. Adipose derived adult stem cells isolation, characterization, and differentiation potential. Cytotherapy 2003;5:362.  Back to cited text no. 6
    
7.
Miranville A, Heeschen C, Sengenès C, Curat CA, Busse R, Bouloumié A. Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation 2004;110:349-55.  Back to cited text no. 7
    
8.
Mehlhorn AT, Niemeyer P, Kaschte K, Muller L, Finkenzeller G, Hartl D, et al. Differential effects of BMP-2 and TGF-beta1 on chondrogenic differentiation of adipose derived stem cells. Cell Prolif 2007;40:809-23.  Back to cited text no. 8
    
9.
Jin XB, Sun YS, Zhang K, Wang J, Ju XD, Lou SQ. Neocartilage formation from predifferentiated human adipose derived stem cells in vivo. Acta Pharmacol Sin 2007;28:663-71.  Back to cited text no. 9
    
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Wu L, Wu Y, Lin Y, Jing W, Nie X, Qiao J, et al. Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix. Mol Cell Biochem 2007;301:83-92.  Back to cited text no. 10
    
11.
Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringdén O. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003;57:11-20.  Back to cited text no. 11
    
12.
Li X, Lee JP, Balian G, Greg Anderson D. Modulation of chondrocytic properties of fat-derived mesenchymal cells in co-cultures with nucleus pulposus. Connect Tissue Res. 2005;46:75-82.  Back to cited text no. 12
    
13.
Kuo YR, Chen CC, Goto S, Lee IT, Huang CW, Tsai CC, et al. Modulation of immune response and T-cell regulation by donor adipose-derived stem cells in a rodent hind-limb allotransplant model. Plastic and Reconstructive Surgery.2011;12:661–672.  Back to cited text no. 13
    
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Gomes ME, Bossano CM, Johnston CM, Reis RL, Mikos AG.In vitro localization of bone growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and cultured in a flow perfusion bioreactor. Tissue Eng 2006;12:177-88.  Back to cited text no. 14
    
15.
Iwashina T, Mochida J, Sakai D, Yamamoto Y, Miyazaki T, Ando K, et al. Feasibility of using a human nucleus pulposus cell line as a cell source in cell transplantation therapy for intervertebral disc degeneration. Spine (Phila Pa 1976) 2006;31:1177-86.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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