Download Human Pluripotent Stem Cell-derived Articular Chondrocytes

Human Pluripotent Stem Cell-derived Articular Chondrocytes Generate and
Maintain Stable Articular Cartilage in vitro and in vivo
April M. Craft, PhD1, Jason Rockel, PhD2, Yulia Nartiss1, Benjamin Alman, MD3, Rita Kandel, MD4, Gordon Keller, PhD1.
1
University Health Network, Toronto, ON, Canada, 2Hospital for Sick Children, Toronto, ON, Canada, 3Duke University, Durham,
NC, USA, 4Mount Sinai Hospital, Toronto, ON, Canada.
Disclosures:
A.M. Craft: None. J. Rockel: None. Y. Nartiss: None. B. Alman: None. R. Kandel: None. G. Keller: None.
Introduction: Osteoarthritis (OA) is a progressively debilitating disease that primarily affects the articular cartilage of synovial
joints. Cell and/or cartilage replacement is a promising therapy for OA, provided that there is access to appropriate tissue and/or
sufficient numbers of articular chondrocytes. Human pluripotent stem cells (hPSCs), including both embryonic stem cells (ESCs)
and induced pluripotent stem cells (iPSCs), represent a novel and potentially unlimited source of chondrocytes and tissues as
these cells are able to generate a broad spectrum of cell types under appropriate conditions in vitro.
Methods: Our approach to achieve efficient development of chondrocytes from PSCs relies on the stage specific recapitulation
of embryonic signaling pathways in serum-free differentiation cultures. Chondrocytes are of mesoderm origin, deriving from
both paraxial and lateral plate mesoderm. Mesoderm arises in the developing embryo during gastrulation, the time at which
epiblast cells migrate through an area of the embryo termed the primitive streak. Studies in our lab have shown that this early
stage of development can be recapitulated in hPSC cultures in vitro by modulating the Activin (nodal), Wnt, and BMP signaling
pathways, thus allowing for the induction of specific mesoderm subsets from hPSCs in an efficient manner. To generate an
appropriate primitive streak-like population, hPSCs were differentiated as embryoid bodies (EBs) in the presence or absence of
recombinant proteins activin, BMP4, basic (b)FGF, and the small molecule inhibitor of GSK3β, CHIR99021 for two to three days.
EBs were then dissociated and cultured as monolayers in the presence or absence of bFGF and a BMP inhibitor for a period of up
to 12 days. The potential of mesoderm subsets was monitored quantitatively by flow cytometry and by the expression of
paraxial/chondrogenic mesoderm-specific transcription factors. To generate cartilage tissues, hPSC-derived chondrogenic
mesoderm was cultured at high density (such as micromass) in the presence or absence of TGFβ superfamily signaling
molecules. hPSC-derived cartilage tissues were characterized histologically and by the expression of cartilage-specific genes. To
determine the in vivo potential of hPSC-derived chondrocytes, micromass-derived tissues were dissociated and chondrocytes
were injected subcutaneously into immunodeficient mice.
Results: We have found that hPSC-derived paraxial/chondrogenic mesoderm, marked by the expression of cell surface receptors
commonly found on adult mesenchymal stem cells, is specified from an activin, Wnt, and BMP induced primitive streak-like
population by inhibition of BMP and addition of bFGF, while the development of closely related cardiac mesoderm was
successfully inhibited in these conditions. Serum-free culture of hPSC-derived chondrogenic mesoderm at high density
(micromass) resulted in the generation of cartilage-like tissues that were ~20 cell layers thick (up to 2mm), and expressed
chondrocyte transcription factors (Sox5, 6, 9) and extracellular matrix components collagen 2 and aggrecan. When cultured in
the presence of appropriate signaling molecules, hPSC-derived chondrogenic mesoderm could be induced to generate cartilagelike tissues with distinct articular cartilage or growth plate (hypertrophic chondrocyte-containing) cartilage phenotypes (Figure
1a). hPSC-derived articular cartilage-like tissues expressed high levels of PRG4 (lubricin; Figure 1b) that were similar to those
found in primary adult human articular cartilage. In addition, these tissues expressed interzone/articular cartilage markers CILP2,
COL1A1, GDF5, WNT9A, and ERG. hPSC-derived growth plate-like cartilage tissue consisted of hypertrophic chondrocytes that
expressed markers indicative of this cell type including COL10A1, RUNX2, SP7, and ALPL. These cells did not express genes
associated with interzone cells or superficial zone chondrocytes. hESC-derived articular and hypertrophic chondrocytes also
generated proteoglycan-rich cartilage tissues in vivo when transplanted into immunodeficient mice (Figure 1c). After 8 and 12
weeks, grafts from hESC-derived growth plate chondrocyte-like cells contained calcified cartilage and/or bone areas suggesting
that these cells/tissues were able to initiate the endochondral ossification process, however, no calcification was observed in
grafts from hESC-derived articular cartilage-like cells at any time point (Figure 1d), indicating that these cells were able to
maintain a stable cartilage phenotype in vivo.
Discussion: These findings demonstrate that through stage-specific manipulation of appropriate signaling pathways it is possible
to efficiently and reproducibly generate distinct populations of hPSC-derived articular or hypertrophic chondrocytes capable of
generating cartilage-like tissues in vitro. Importantly, we observed that the hPSC-derived articular chondrocytes maintained an
articular cartilage-like phenotype over a 12 week period in vivo.
Significance: Access to hPSC-derived cartilage tissues will be instrumental for the development of cell and tissue based
engineering strategies for articular cartilage replacement in addition to other joint and bone disorders. Additionally, the ability
to generate such tissues will facilitate applications such as modeling cartilage disorders in vitro using patient-specific iPSCs and
identifying pathways and/or drugs that may be important for potentiating or attenuating OA.
Acknowledgments: CIHR, McEwen Centre for Regenerative Medicine, Krembil Foundation
ORS 2014 Annual Meeting
Poster No: 1183