The central question for engineering a native-like tissue is firstly to identify and define the ‘ideal’ stem or progenitor cell type for tissue regeneration, and secondly the ‘cell source’ that can be used to derive such a progenitor cell. Although the tissue-resident stem cell-mediated regenerative processes are well defined and understood in molecular details for tissues like muscle, skin, and bone, cartilage regeneration or lack thereof remains enigmatic. Multiple studies have explored the presence of a stem/progenitor-like population in cartilage [30] and there is some recent evidence for the existence of small stem-like populations marked by growth differentiation factor 5 (Gdf5) [31] or lubricin (PRG4) [32]. However, it remains a clinical reality that these cell populations are unable to effectively regenerate into functional cartilage, unlike bone or muscle [1]. Because of the lack of a consensus cartilage stem cell, cell sources that are utilized for cartilage transplantation in the clinic are limited to autologous chondrocytes. Techniques like microfracture are utilized to provide access for endogenous bone-marrow mesenchymal stem cells (MSCs) to cartilage; however, the MSCs at best give rise to functionally suboptimal fibrocartilage, due to its lack of t native collagen ECM. Tissue engineering approaches have explored alternative stem cell sources including adipose-derived stem cells (ADSCs) [33], clonal blood marrow precursors [34], and nasal cartilage-derived progenitor cells [35]; however, the persistent paucity of cells and a rapid loss of stemness upon expansion has been a bottleneck for these cell sources as well.

Previous studies have suggested that juvenile chondrocytes could be an attractive cell source for cartilage generation owing to their faster proliferation and expansion, but their widespread use is limited by donor availability. Patient-specific hiPSCs can be generated from readily available cells including blood and skin cells, thereby overcoming the major bottleneck of the paucity of cells and donor availability. The aim of these studies was to provide a detailed side-by-side phenotypic as well as molecular comparison of adult, juvenile, and human iPSC-derived chondrocytes (hiChondrocytes). hiChondrocytes were generated from hiPSCs using our previously published methodology that led to an efficient and homogeneous differentiation [18]. hiChondrocytes exhibited a higher proliferation rate similar to juvenile chondrocytes that would be advantageous in their expansion for research or therapeutic applications.

Detailed analyses of global gene expression patterns of hiChondrocytes, juvenile chondrocytes, and adult chondrocytes allowed us to dissect the common and distinct molecular factors between the chondrocyte populations. We expected variability in the chondrocyte populations isolated from different donors (i.e., juvenile and adult chondrocytes as well as different hiChondrocyte lines). Studies such as those reported here have the caveat that they are based on a small subset of available samples. Analyses of large cohorts of ‘normal’ juvenile or adult chondrocytes are a challenge due to both the limited availability of these samples and the limited cell expansion possible before these cells dedifferentiate in culture. These studies, however, are useful in identifying putative differential ‘factors’ and their functions that can be potentially utilized for modulating and rejuvenating available chondrocyte populations. For example, our analyses report novel ECM genes (CHRDL1 and MFAP4) that are enriched in both juvenile and hiChondrocytes as compared to the adult chondrocytes. One of these ECM genes, CHRDL1, is stimulatory for resident MSCs [21] and could therefore contribute to a superior regeneration in vivo.

Another surprising find was that immune response pathways were differentially enriched both in hiChondrocytes and in juvenile chondrocytes. This led to the interesting finding that hiChondrocytes mimic juvenile chondrocytes in a milder response to proinflammatory cues compared to adult chondrocytes. Upon IL-1β stimulation, both hiChondrocytes and juvenile chondrocytes showed a lower upregulation of inflammatory (like CCL2 and IL6) and catabolic (MMP3 and ADAMTS4) genes than adult chondrocytes. In addition, hiChondrocytes and juvenile chondrocytes were also resistant to dedifferentiation compared to adult chondrocytes since they maintained the expression of chondrogenic genes, COL2A and SOX9, in the presence of IL-1β, while these chondrogenic genes were rapidly downregulated in adult chondrocytes.

CD24 is a small, glycosyl-phosphatidylinositol (GPI)-anchored cell-surface protein that is heavily glycosylated in a highly tissue and context-dependent manner. CD24 lacks an intracellular domain but is known to interact with multiple partners leading to different signaling outcomes in neural and hematopoietic tissues, including effects on proliferation, differentiation, and inflammation [36, 37]. CD24 was previously found to be enriched in juvenile chondrocytes compared to adult chondrocytes and was reported by us to be protective against IL-1β [26]. In the present studies, we have also identified its effect on chondrocyte proliferation since a loss of CD24 led to reduced cell proliferation. CD24 is therefore one of the factors that contribute to the dual advantage of both higher proliferation and resistance to IL-1β in hiChondrocytes and juvenile chondrocytes, characteristics that could potentially translate into superior cartilage regeneration in vivo.

Upon encapsulation of chondrocytes in chondroitin sulfate containing polyethylene glycol (CS-PEG)-based biomimetic hydrogels, transplantation in vivo in immune-deficient mice, and harvesting after 4 weeks, the cartilage constructs generated by hiChondrocytes and juvenile chondrocytes showed high expression of distinct chondrogenic genes (i.e., SOX9, COL2A1, AGAN) and minimal or no expression of the hypertrophy marker COl10A1. These data are consistent with previously established quantitative functional methods to distinguish juvenile and adult chondrocytes with characteristics such as increased ECM production by the juvenile chondrocytes compared to adult chondrocytes [20]. Importantly, these data suggest that hiChondrocytes are similar to juvenile chondrocytes in their high proliferative capacity and generation of cartilage constructs in vivo in hydrogel scaffolds. Although our studies had to be carried out in immune-deficient mice to prevent outright rejection of the human cells in mice, it can be imagined that the resistance to IL-1β would likely be a further big advantage in the utility of hiChondrocytes in a clinical setting in diseases like osteoarthritis or rheumatoid arthritis where end-stage chronic inflammation is a major component.