Effects of Culturing on the Stability of the Putative Murine Adipose Derived Stem Cells Markers

Abstract

Mesenchymal stem cells have generated much interest because of their potential use in regenerative medicine. The major draw back in the application of these cells is that there is no single marker or markers that have been established to identify and aid in isolating the cells from a variety of other cell types. The commonly expressed mesenchymal stem cell surface antigens include CD44, CD73, CD90.2, CD105, and CD146. In the present study we examined the stability of these surface antigens in culture and their potential application in identifying and isolating murine derived adipose derived stem cells. The data showed that the expression of these markers increased with culturing and appeared to stabilize by passage 8; the cells were sorted positively for the surface markers at this passage. Each subset was maintained in culture and evaluated for differentiation toward osteogenic lineage in vitro and in vivo. The CD73 and CD105 positive cell subsets demonstrated robust differentiation toward osteogenic lineage in vitro; the CD90.2+ cell subset exhibited the least differentiation toward osteogenic lineage. Assessment of the cell subpopulations for in vivo differentiation demonstrated that all the cell subsets exhibited potential to differentiate into osteoblasts. Taken together, these data suggest that this panel of markers although useful in identifying cells with potential to differentiate toward osteogenic lineage, cannot prospectively be used for enriching for ADSC from a variety of other cell types.

Fig. 1. Morphological appearance of the unsorted ADSCs isolated from the murine inguinal fat pad

(A) The cells are shown under bright field and under fluorescence imaging for GFP (scale bars are 200μm). (B) Differentiation of the Unsorted ADSCs; ALP activity after BMP-2 treatment, Alizarin Red S staining of the cells after 21 days in osteogenic medium, Oil Red O staining of the cells maintained in culture for 14 days in adipogenic medium. BMP-2 treatment induced increase in ALP activity. The cells show differentiation toward osteogenic and adipogenic cell lineages in vitro.

Fig. 2. Expression of selected surface antigens by the sorted ADSCs during expansion in culture

(A) The panel of putative murine MSC markers analyzed by flow cytometry during culturing from Passage 3 to Passage 10 are shown. The percentage of cells in the total population expressing each marker is noted below each histogram. Note the changes in marker expression during passaging. (B) Changes of the antigen expression during cell expansion analyzed by flow cytometry in the graphical form. CD73 and CD146 expression increased with passaging; CD44, CD105 stayed constant during culturing; CD 90.2 remained low throughout the expansion period with a slight decrease during expansion in culture.

Fig. 3. Morphological appearance and stability of the sorted cell subsets

The unsorted, CD73+ and CD105+ cells appear to exhibit similar morphologies because cells expressing these antigens are predominant in the preparation. The CD90.2+ subsets display a different morphology because they represented a smaller population of the cells in the preparation. The cells expressing CD73 also express CD105. Because these cells are predominant in the preparation they exhibit similar morphologies with unsorted cells. (scale bars are 200μm). CD90.2+ cells appear to comprise cells that are smaller with a round morphological appearance whereas the other cell subsets appear spindle shaped.

Fig. 4. Osteogenic differentiation of the sorted cell subsets in vitro

(A) ALP activity of each cell subset. The Unsorted, CD105+ and CD73+ showed similar level of ALP activity. The CD90.2+ cells showed reduced ALP activity expression. (B) Alizarin Red S staining of the same cell subsets for calcium deposition (scale bars are 300μm). The Unsorted, CD105+ and CD73+ cells show extensive staining for calcium deposition. The CD90.2+ cells show reduced staining for calcium confirming the ALP activity data. The basal ALP activity of CD73 is lower than the other cell subsets suggesting that the other cell subsets might contain cells that already express higher levels of ALP activity.

Fig. 5. Expression of the osteoblast differentiation genes at different time points by each of the donor cell subset retrieved from the femurs infused with the cells

(A) Representative morphological appearance of ADSCs retrieved from the bone and bone marrow. (B) Before infusion of the cell subsets into femurs, all the cell subsets analyzed expressed osteopontin constitutively. At 1 week, all the donor cell subsets retrieved from bone and marrow expressed Runx2. At 2 weeks, the donor cell subsets retrieved from bone and marrow continued to show expression of Runx2. At 4 weeks, expression of osterix is seen as well as osteoclacin. Osteocalcin is expressed by cells retrieved from bone, not marrow. The donor cells were retrieved from the recipient femurs at the indicated time points, cultured in presence of Zeocin and assessed for expression of the indicated genes. B= bone, M= marrow, Un= unsorted cells.