Year 1

The second year of the study is focused on identifying the molecular mechanisms of (Ca)/calmodulin-dependent protein kinase (CaMKII) in regulating the chondrogenic differentiation of human pluripotent stem cell-derived chondrocytes/chondrospheres (hPDCs) and to determine whether the inhibition of CaMKII activity prevents hypertrophy of hPDCs. CaMKII (also known as CaMK2) isoforms assemble into homo or heteromultimeric holoenzymes composed of 8 to 12 subunits. Four CaMKII genes (α, β, γ and δ) in humans give rise to 40 isoforms through alternative splicing. CaMKII is a Serine/Threonine protein kinase that is activated by Ca2+/CaM binding. CaMKII activity is up-regulated prior to hypertrophy and loss of CaMKII function substantially blocks the transition from proliferation to hypertrophy. Our findings from this reporting period demonstrated that PSC-derived cartilage cells closely resemble normal cartilage developmental program. Terminal differentiation and hypertrophy of PSC-derived chondrocytes can be inhibited by either pharmacological or genetic inhibition of CAMKII signaling. This finding indicates that inhibition of CAMKII signaling may be beneficial for generation of the phenotypically stable cartilage tissue for joint repair

Our previous studies described in Year 1 report predicted that adenosine and its receptors (A1, A2a, A2b, A3) play an important role in regulating homeostasis within the synovial joint. Previous literature has demonstrated that adenosine is produced within the synovial cavity by specific cell type – synovial pericytes. This population of pericytes specifically expresses cell surface markers CD73 and CD39. During this year we tested whether adenosine signaling plays a role in regulating cartilage differentiation via CaMKII signaling. Our results indicate that adenosine normally produced by synovial pericytes plays a critical role in regulation of cartilage differentiation and CaMKII signaling. Stimulation of adenosine receptors 1 and 3 with small molecule agonists dramatically inhibited activation of CaMKII and differentiation of PSC-derived chondrocytes nominating these regulators as potential candidates for tissue engineering approaches aiming to produce phenotypically stable cartilage tissue.

Next year we will test applicability of the above findings in vivo to assess whether better hyaline cartilage can be generated. Long term goal of this work is to produce high quality hyaline cartilage replacement for patients with osteoarthritis