The Experimental Study Of Beagle Dog Adipose-derived Stem Cells Differentiating Toward Nucleus Pulposus-like Cells

In modern society, with the increased aging of population and work pressure, there aremore and more people who suffer the low back pain. Degenerative disc disease (DDD)is one of the major causes of low back pain. DDD has serious impact on the daily life,and it imposes the economic loan in family and the society. Some experimental studyshowed that the disc degeneration began with nucleus pulposus（NP）, the number ofNP cells reduced and extracellular matrix components changed. Current clinicaltreatments are symptomatic rather than curative, and the long-term effect is not good.With the development of medical technology, cell transplantation or tissue engineeringnucleus pulposus offers a treatment that both cures the problem of DDD and restoresnormal disc function. One of the major problems is to select the ideal seed cells source.Adipose-derived stem cells (ADSCs) which have the advantages of easier obtained,low trauma，faster proliferation and stable property have been used in regenerationresearch. Additionally, previous studies have shown that ADSCs can differentiate intoNP-like cells through the co-culture of ADSCs and NP cells, and many growth factorsplay a vital role in the process such as TGF-β. At present, the experimental study ofBeagle dog ADSCs differentiating into NP-like cells with TGF-β1and BMP-7wereless reported.In our study, drawing on the results of previous studies of biological therapy, we willselect the Beagle dog ADSCs as the seed cells. We investigate whether ADSCs candifferentiate into NP-like cells induced by TGF-β1and BMP-7. We hope it can provide experimental basis for seed cells transplantation and tissue engineering therapy in thetreatment of intervertebral disc degeneration.Objectives: To isolation, culture the Beagle dog ADSCs, observe the cell morphologyand biological characteristics, and test the surface markers of mesenchymal stem cells.To research ADSCs proliferate in the culture condition containing TGF-β1、BMP-7,and verify whether ADSCs can differentiate into NP-like cells.Methods: ADSCs were obtained from Beagle dog fat removed from inguinal region.At80%confluence, cells were trypsinized and re-seeded. The morphology change ofADSCs was observed by inverted microscope. ADSCs at passage3were used forgrowth curve. Three passaged ADSCs were analyzed by flow cytometry for surfacemolecule such as CD34、CD45、CD73、CD105、HLA-DR、HLA-ABC. Then ADSCswere cultured in the6-well culture plate at1×105/ml cells, divide them into two groups.The experimental group was cultured in a special medium with TGF-β1and BMP-7,and the control group was cultured in the ordinary culture medium. The ability ofproliferation was assessed by CCK-8. After7,14days, the expression mRNA of type Icollagen, type II collagen and aggrecan were analysed by Real Time-PCR.Results: The primary ADSCs were partly attachment at2-3day in spindle-shaped, andthe cells gradually spread out at the6-7day. After10days, cells had grown to80%confluent with fibroblast-like morphology. The growth curve of ADSCs presented the“S” shape. Flow cytometry analysis showed that the ADSCs were positive for CD73、CD105、HLA-ABC, and negative for CD34、CD45、HLA-DR. The experimental groupcells were changed to polygon after7days. CCK-8analysis showed the proliferationof experimental group cells was impacted. At the7day, Real Time-PCR demonstratedthat type II collagen and aggrecan mRNA expression were up regulated (p<0.05). While there were no significant changes in type I collagen mRNA expression betweentwo groups（P>0.05）. At the14day，the gene expression levels of type II collagen alsohad significantly increase in the experimental group（P<0.05）, but it was lower thanthat of7day. There was no significant change in the gene expression levels ofaggrecan and type I collagen between two groups.Conclusion：In our study, ADSCs were isolated from the Beagle dog fat with type Icollagen enzyme digestion. ADSCs could proliferate rapidly in vitro and express thesurface markers of MSCs, didn’t express the hematopoietic lineage markers. But thespecial culture medium inhibited the proliferation of ADSCs. Our results indicate thatTGF-β1and BMP-7can drive ADSCs differentiation towards a phenotype consistentwith that of NP cells. ADSCs might be a promising seed cell source, and this study laysa foundation for tissue engineering and cell-based transplantation therapy ofintervertebral disc degeneration.