Preliminary Studies Of Chondrogenic Potential Of Human Adipose-Derived Stem Cells Sorted For CD34~+ And CD31~- In Vitro

BackgroundTissue engineering is a promising treatment modality in treatment for cartilage defect, but there are still problems with the choice of seed cells. Adipose-derived stem cells (ASCs) are able to differentiate into various cell types of the mesodermal lineage conditions in vitro. The cells can be easily isolated by liposuction with relatively low morbidity and pain. Also they are abundant in adipose tissue, which is easily accessible. For these reasons, ASCs are attractive sources for cartilage tissue engineering. However, the stromal vascular fraction (SVF) obtained from adipose tissue is a heterogeneous population of mononuclear cells that includes relative low fraction of mesenchymal stromal cells, and show an insufficient potential for chondrogenesis. As a result, we need to sort the SVF to acquire purified ASCs. It has been demonstrated that freshly isolated ASCs are CD34 positive and CD31 negative, thus we use flow cytometry to sort ASCs for CD34+and CD31-, and to characterize the chondrogenic potential of ASCs(CD34+CD31-).ObjectivesIn our study, CD34+CD31- population within the SVF were sorted and the surface markers to identify whether the population was mesenchymal stem cells. The chondrogenic potential of sorted CD34+CD31- population was compared with the SVF, to investigate whether the CD34+CD31- subset is the ideal seed cell for cartilage tissue engineering.Methods and Results1. Isolation and phenotype identification of the ASCs (CD34+CD31-) Methods:The SVF was obtained from lipoaspirate digested with collagenase type Ⅰ, and then sorted for CD34+CD31- subset by flow cytometry. The mesenchymal stem cell smarkers were identified for both the sorted CD34+CD31’subset and SVF.Results:About 88.37% of the SVF express CD34. The CD34+CD31- subset account for 66.74% of the SVF, and the ratio increased with aging. The sorted CD34+CD31-subset coexpressed CD44, CD90, CD73, and did not express CD45, CD105. The expression of CD44, CD90, CD73 was higher in the CD34+CD31- population than that in the SVF.2. Assessment of the chondrogenic differentiation capacity of hASCs (Human adipose-derived stem cells) (CD34+CD31-) in plate cultureMethods:The sorted hASCs (CD34+CD31-) and SVF were seeded in plates containing chondrogenic medium. The expression of chondrogenic gene aggrecan, collagen Ⅱ and chondrogenic trophic gene collagen Ⅹ were analyzed by Real-time PCR. Cartilage proteoglycan link protein, and collagen type Ⅱ were assessed by toluidine blue staining and collagen Ⅱ immunostaining.Results:The expression of aggrecan, collagen type Ⅱ and collagen type Ⅹ were upregulated in both hASCs(CD34+CD31-) and SVF after 14 days of chondrogenesis culture, but no statistic differences show between the two groups. Toluidine blue staining and collagen Ⅱ immunostaining showed similar chondrogenesis in two groups.3. Assessment of the chondrogenic differentiation capacity of hASCs (CD34+CD31-) in pellet cultureMethods:The hASCs (CD34+CD31-) and SVF were cultured in pellets for chongdrogenesis. The specimens were compared by gross inspection, histology, immunohistochemistry, and quantitative GAG assays.Results:Cartilage differentiation was better in hASCs (CD34+CD31-),confirmed by gross inspection, toluidine blue and Safranin-O staining and immunohistochemistry. GAG assays demonstrated no statistic difference between two groups.Conclusions1. CD34 was highly expressed in SVF, and the ratio of SVF which express CD34 did not change with age. The proportion of CD34+CD31- population increased with age. The expression of stem cell markers was higher in the CD34+CD31- subset than that in SVF, indicating that the sorted CD34+CD31’cells were ASCs.2. In monolayer culture, hASCs (CD34+CD31-) can differentiate into chondrogenic cells, but with no better capacity compared with SVF.3. In pellet culture, hASCs (CD34+CD31-) displayed superior potential in chondrogenesis compared with SVF in gross inspection, histology and immunohistochemistry, which suggests that the hASCs (CD34+CD31-) may be better than SVF as seed cells.