Jose Diaz-Romero

Immunophenotypic analysis of human articular chondrocytes: Changes in surface markers associated with cell expansion in monolayer culture

Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non‐adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as “dedifferentiation.” To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post‐mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up‐regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications.

Immunophenotypic changes of human articular chondrocytes during monolayer culture reflect bona fide dedifferentiation rather than amplification of progenitor cells

In this study, a time‐course comparison of human articular chondrocytes (HAC) and bone marrow‐derived mesenchymal stem cells (MSC) immunophenotype was performed in order to determine similarities/differences between both cell types during monolayer culture, and to identify HAC surface markers indicative of dedifferentiation. Our results show that dedifferentiated HAC can be distinguished from MSC by combining CD14, CD90, and CD105 expression, with dedifferentiated HAC being CD14+/CD90bright/CD105dim and MSC being CD14‐/CD90dim/CD105bright. Surface markers on MSC showed little variation during the culture, whereas HAC showed upregulation of CD90, CD166, CD49c, CD44, CD10, CD26, CD49e, CD151, CD51/61, and CD81, and downregulation of CD49a, CD54, and CD14. Thus, dedifferentiated HAC appear as a bona fide cell population rather than a small population of MSC amplified during monolayer culture. While most of the HAC surface markers showed major changes at the beginning of the culture period (Passage 1–2), CD26 was upregulated and CD49a downregulated at later stages of the culture (Passage 3–4). To correlate changes in HAC surface markers with changes in extracellular matrix gene expression during monolayer culture, CD14 and CD90 mRNA levels were combined into a new differentiation index and compared with the established differentiation indices based on the ratios of mRNA levels of collagen type II to I (COL2/COL1) and of aggrecan to versican (AGG/VER). A correlation of CD14/CD90 ratio at the mRNA and protein level with the AGG/VER ratio during HAC dedifferentiation in monolayer culture validated CD14/CD90 as a new membrane and mRNA based HAC differentiation index. J. Cell. Physiol. 214:75–83, 2008.

Population doublings and percentage of S100‐positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57–4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90‐negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90‐positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100‐positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell‐based cartilage repair treatments. J. Cell. Physiol. 222: 411–420, 2010.

S100A1 and S100B expression patterns identify differentiation status of human articular chondrocytes.

Many studies in the field of cell‐based cartilage repair have focused on identifying markers associated with the differentiation status of human articular chondrocytes (HAC) that could predict their chondrogenic potency. A previous study from our group showed a correlation between the expression of S100 protein in HAC and their chondrogenic potential. The aims of the current study were to clarify which S100 proteins are associated with HAC differentiation status and to provide an S100‐based assay for measuring HAC chondrogenic potential. The expression patterns of S100A1 and S100B were investigated in cartilage and in HAC cultured under conditions promoting dedifferentiation (monolayer culture) or redifferentiation (pellet culture or BMP4 treatment in monolayer culture), using characterized antibodies specifically recognizing S100A1 and S100B, by immunohistochemistry, immunocytochemistry, Western blot, and gene expression analysis. S100A1 and S100B were expressed homogeneously in all cartilage zones, and decreased during dedifferentiation. S100A1, but not S100B, was re‐expressed in pellets and co‐localized with collagen II. Gene expression analysis revealed concomitant modulation of S100A1, S100B, collagen type II, and aggrecan: down‐regulation during monolayer culture and up‐regulation upon BMP4 treatment. These results strongly support an association of S100A1, and to a lesser extent S100B, with the HAC differentiated phenotype. To facilitate their potential application, we established an S100A1/B‐based flow cytometry assay for accurate assessment of HAC differentiation status. We propose S100A1 and S100B expression as a marker to develop potency assays for cartilage regeneration cell therapies, and as a redifferentiation readout in monolayer cultures aiming to investigate stimuli for chondrogenic induction. J. Cell. Physiol. 229: 1106–1117, 2014.

Hierarchical clustering of flow cytometry data for the study of conventional central chondrosarcoma

We have investigated the use of hierarchical clustering of flow cytometry data to classify samples of conventional central chondrosarcoma, a malignant cartilage forming tumor of uncertain cellular origin, according to similarities with surface marker profiles of several known cell types. Human primary chondrosarcoma cells, articular chondrocytes, mesenchymal stem cells, fibroblasts, and a panel of tumor cell lines from chondrocytic or epithelial origin were clustered based on the expression profile of eleven surface markers. For clustering, eight hierarchical clustering algorithms, three distance metrics, as well as several approaches for data preprocessing, including multivariate outlier detection, logarithmic transformation, and z‐score normalization, were systematically evaluated. By selecting clustering approaches shown to give reproducible results for cluster recovery of known cell types, primary conventional central chondrosacoma cells could be grouped in two main clusters with distinctive marker expression signatures: one group clustering together with mesenchymal stem cells (CD49b‐high/CD10‐low/CD221‐high) and a second group clustering close to fibroblasts (CD49b‐low/CD10‐high/CD221‐low). Hierarchical clustering also revealed substantial differences between primary conventional central chondrosarcoma cells and established chondrosarcoma cell lines, with the latter not only segregating apart from primary tumor cells and normal tissue cells, but clustering together with cell lines from epithelial lineage. Our study provides a foundation for the use of hierarchical clustering applied to flow cytometry data as a powerful tool to classify samples according to marker expression patterns, which could lead to uncover new cancer subtypes. J. Cell. Physiol. 225: 601–611, 2010.

S100A1 and S100B: Calcium Sensors at the Cross‐Roads of Multiple Chondrogenic Pathways

The S100 protein family comprises more than 20 members of small calcium binding proteins operating as Ca2 +‐activated switches that interact and modulate the activity of a large number of targets. S100A1 and S100B, two members of this family, have been recently associated with the differentiation status of human articular chondrocytes. Both proteins are homogeneously expressed in all cartilage zones, their expression decreases during chondrocyte dedifferentiation, and can be induced under conditions promoting redifferentiation. Although S100 proteins have a broad range of extra‐ and intracellular roles, functional studies of S100 proteins expressed in chondrocytes have focused on their extracellular roles linked to catabolic processes. The intracellular roles of S100A1 and S100B in chondrocytes remain largely unexplored, yet the few studies addressing their intracellular activity point toward potentially important functions in chondrocyte biology. This review summarizes reported intracellular S100A1 and S100B regulatory functions described in other cell types that could be also involved in the regulation of chondrogenic processes in cartilage. Potential roles of S100A1 and S100B in the TGF‐β‐SMAD, the cAMP–PKA–CREB, and the PI3K‐AKT pathways, Ca2+ homeostasis, cytoskeleton dynamics, the calcineurin‐NFAT pathway, interactions with the p53 family, and the Hippo pathway are examined in the context of chondrocyte biology. Based on the plethora of interactions of S100A1 and S100B with different molecular partners playing essential roles in chondrocyte biology, and the staggering complexity and ubiquity of cross‐talk among these partners, we hypothesize that these S100 proteins play fundamental roles in the spatial and temporal regulation of chondrogenesis. J. Cell. Physiol. 232: 1979–1987, 2017.

S100B + A1 CELISA: A Novel Potency Assay and Screening Tool for Redifferentiation Stimuli of Human Articular Chondrocytes.

During monolayer expansion, a necessary step in autologous chondrocyte implantation, human articular chondrocytes (HAC) dedifferentiate and lose their capacity to produce stable hyaline cartilage. Determining HAC potency and learning how to trigger their redifferentiation would improve cell‐based cartilage regeneration therapies. We previously identified S100B and S100A1 proteins as markers of HAC redifferentiation potential. Here, we aimed to: (i) demonstrate a correlation between S100B + A1‐positive HAC in monolayer culture and their neochondrogenesis capacity in pellet culture; (ii) develop an S100B + A1 cell‐based ELISA, and (iii) prove that S100B + A1 induction in HAC increases their chondrogenic capacity. Expression patterns of S100A1 and S100B were investigated in HAC during dedifferentiation (monolayer) or redifferentiation (pellet or high‐osmolarity/BMP4 treatment in monolayer) using qRT‐PCR, immunocytochemistry, or immunohistochemistry. A cell‐based ELISA (CELISA) was developed as a 96‐well microplate multiplex assay to measure S100B + A1 (chondrogenesis), alkaline phosphatase (hypertrophy), and DNA amount (normalization), and applied to HAC, bone marrow‐derived mesenchymal stem cells and the chondrocytic cell line ATDC5. The direct correlation between the percentage of S100B + A1‐positive HAC in monolayer and their neochondrogenesis in pellets validates S100B + A1 as a marker of chondrogenic potency. The S100B + A1‐CELISA accurately determines HAC differentiation status, allows identification of chondrogenic stimuli, and permits the simultaneous monitoring of the undesirable hypertrophic phenotype. This novel assay offers a high‐throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and for identifying redifferentiation factors/conditions. HAC improved neochondrogenesis in pellets—induced with high‐osmolarity and BMP4 treatment in monolayer—suggests that cell instruction prior to implantation may improve cartilage repair. J. Cell. Physiol. 232: 1559–1570, 2017.