Maintenance of "stem cell" features of cartilage cell sub-populations during in vitro propagation

Abstract

Background: The discovery of mesenchymal stem cells (MSCs) or MSC-like cells in cartilage tissue does not tie in well with the established view that MSCs derive from a perivascular niche. The presence of MSCs may raise concerns about specificity and application safety, particularly in terms of the regulatory site. The aim of the present study was to investigate the benefits or possible risks of the MSC-like properties of cells isolated from cartilage in the context of autologous chondrocyte implantation.

Methods: Chondrocytic cells were isolated from cartilage or intervertebral disc tissue. Flow cytometry was used to analyze the expression of cell surface antigens. MSC-like cells were either enriched or depleted by means of magnetic cell sorting (MACS) involving the monoclonal antibodies W5C5/SUSD2 and W8B2/MSCA-1. We addressed the issues of prolonged expansion of such cells as well as the influence of culture medium as a trigger for selecting a single cell type. Established protocols were used to study in vitro differentiation. In addition to histological and biochemical assessment, the acquired phenotypes were also evaluated on the mRNA transcript level.

Results: In the studied cells, we found strongly analogous expression of antigens typically expressed on MSCs, including CD49e, CD73, CD90, CD105, CD140b and CD166. The expression of W5C5 and W8B2 antigens in cartilage cell sub-populations did not correlate with multi-potency. We demonstrated that a chondroid precursor, but not a bona fide multipotent mesenchymal, cell type can be obtained under established in vitro culture conditions. The culture media used for expansion influenced the cell phenotype.

Conclusions: The risk of adverse adipose or osseous differentiation is not posed by expanded chondrocyte cultures, even after enrichment of putative MSC-like cell populations by MACS. It is possible that this limited "stemness" in chondrocytes, expanded for use in ACI, may instead be beneficial as it allows re-differentiation under appropriate conditions despite prolonged times in culture.

Figure 1

Differentiation of chondrocytes and sorted sub-populations. Expression of mRNA in primary expanded human chondroyctes after MACS separation (P0), and subsequent passage in monolayer (P1 ML) or in hydrogel culture (P1 Gel). The P1 ML cells were cultured in chondrocyte medium (CM) until they had reached approx. 80% confluence (about 1 week), hydrogels were maintained in CM for 6 weeks. The expression of collagen type I (COL1), type II (COL2), and aggrecan (ACAN) was analyzed and the ratio of COL2/COL1 was calculated as a measure to describe the chondrogenic phenotype of the cells as indicated. Box plots represent 25% / 75% percentiles, mean values (dotted lines) and median values (hairlines). Lower right: the cumulated glycosaminoglycan production from cells in hydrogel culture was determined. GAG content was measured in the hydrogel and in the combined medium supernatant of the six-week cultures and normalized to the corresponding DNA content in the hydrogel. Mean values and standard deviation are shown (n = 3 cultures).

Figure 2

Cell surface antigen expression of selected markers on cartilage cell populations. Human chondrocytes (P0), and human disc cells (P0) were stained with the indicated phycoerythrin-conjugated monoclonal antibodies. The black lines show the fluorescence intensity of the IgG negative control antibody, whereas the intensity of the antibody of interest is shown in red. Representative analyses are shown.

Figure 3

Cell growth and morphology of chondrocytes and sub-populations. A) Unsorted chondrocytes and W5C5 or W8B2 enriched (+) or depleted (-) sub-populations of the same culture (Cho4) were expanded over several passages. Population doublings and culture time are presented (top). Chondrocytes (Cho8) and W5C5 ± sub-populations are cultured in chondrocyte culture medium (CM) or MSC growth medium (MSCGM). Growth curves are shown (bottom). B) Cell morphology of different sub-populations. (Due to the low expression of W8B2 on chondrocytes, the number of sorted cells was too low for a complete set of experiments).

Figure 4

Gene expression of chondrocytic sub-populations dependent on the population doublings. Primary expanded chondrocytes and disc cells were MACS separated and expanded in monolayer cultures over several passages. Gene expression was determined at the end of the first passage after MACS sorting (MACS P0), when cells had undergone 4 ± 1 pds after MACS, and after cells had undergone more than 8 pds after MACS. Expression of collagen type I (COL1), collagen type II (COL2), and the transcription factors SRY (sex determining region Y)-box 9 (SOX9) and core-binding factor subunit alpha-1 (CBFA1) were analyzed and expressed relative to the reference gene GAPDH. *: significant difference (p < 0.05) between all chondrocyte populations compared to all disc populations. (For mean expression values see Additional file 1: Table S4).

Figure 5

Adipogenic differentiation of sub-populations after > 8 pds. Adipogenic differentiation was induced using adipogenic induction medium, control cells were cultured in MSC growth medium (MSCGM). Three weeks after induction gene expression of the adipogenic markers, the transcription factor peroxisome proliferator-activated receptor γ 2 (PPARγ2) and adiponectin was analyzed. Expression relative to GAPDH is presented for each individual culture (left). In parallel, lipid droplets were visualized by Oil Red O staining (bars = 200 μm). Representative pictures of one culture after induction are shown. Only very sporadically were positive cells found (pictures with higher magnification on the right). None of the control cultures was positive. *: significant difference (p < 0.05) between all control cultures compared to all cultures under induction conditions. (For mean expression values see Additional file 1: Table S5).

Figure 6

Osteogenic differentiation of sub-populations after > 8 pds. Osteogenic differentiation was induced using osteogenic induction medium, control cells were cultured in MSC growth medium (MSCGM). Three weeks after induction gene expression of the osteogenic markers, the transcription factor core-binding factor subunit alpha-1 (CBFA1) and alkaline phosphatase (ALP) were analyzed. Expression relative to GAPDH is presented for each individual culture (top). ALP activity was determined in the cell lysates at the end of the culture and was normalized to the corresponding DNA content in the well. In parallel, mineralization was visualized by Alizarin Red staining (bar = 200 μm). Representative pictures of four induced cultures are shown. None of the control cultures was positive. *: significant difference (p < 0.05) between all control cultures compared to all cultures under induction conditions. (For mean expression values see Additional file 1: Table S6).

Figure 7

Chondrogenic differentiation of sub-populations after > 8 pds. Chondrogenic differentiation was performed in hydrogels and induced with TGFβ₃, control cells were cultured in hydrogels in the same medium without TGFβ₃. Four weeks after induction gene expression of the chondrogenic markers, the transcription factor SRY (sex determining region Y)-box 9 (SOX9), collagen type II (COL2) was analyzed and compared to the expression of the cells before being embedded into the hydrogels (ML > 8pds). Expression relative to GAPDH is shown for each individual culture (top). Quantification cycles (C_q) for GAPDH expression are shown in the box plot. GAG synthesis (GAG content of the hydrogel + content in the cumulated culture supernatants) is presented as normalized to the DNA content (lower right). Boxes represent 25% / 75% percentiles, whisker 5% / 95% percentiles, mean (dotted line) and median (solid line) values. *: significant difference (p < 0.05) between all ML > 8 pds cultures compared to all gel control cultures and compared to gel cultures under induction conditions. (For mean expression values see Additional file 1: Tables S7 and S8).

Figure 8

Comparison of chondrocyte sub-populations as a function of population doublings. Cells were analyzed at the end of the first passage after MACS and subsequent expansion until cells had undergone more than 8 population doublings (pds). A) Flow cytometry analysis with percentage of W5C5 positive cells after W5C5 MACS sorting and W8B2 positive cells after W8B2 MACS sorting. Representative examples are shown. B) Adipogenic differentiation of subpopulations. Adiponectin expression relative to GAPDH. C) Osteogenic differentiation. ALP gene expression relative to GAPDH and ALP enzyme activity were presented. Boxes represent 25% / 75% percentiles, whisker 5% / 95% percentiles, mean (dotted line) and median (solid line) values. (n = 3 donors after MACS, n = 4 donors + > 8pds). *: significant differences (p < 0.05) between cells differentiated directly after MACS and after expansion, and between subpopulations under the same experimental conditions. Significant differences existed in all cases between the control and the induction conditions (not specifically indicated).