Differences in the intrinsic chondrogenic potential of human mesenchymal stromal cells and iPSC-derived multipotent cells.

BACKGROUND: Human multipotent progenitor cells (hiMPCs) created from induced pluripotent stem cells (iPSCs) represent a new cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) are needed to enhance transforming growth factor-ß (TGFß)-induced hiMPC chondrogenesis. In contrast, TGFß alone is sufficient to result in robust chondrogenesis of human primary mesenchymal stromal cells (hMSCs). Currently, the mechanism underlying this difference between hiMPCs and hMSCs has not been fully understood. METHODS: In this study, we first tested different growth factors alone or in combination in stimulating hiMPC chondrogenesis, with a special focus on chondrocytic hypertrophy. The reparative capacity of hiMPCs-derived cartilage was assessed in an osteochondral defect model created in rats. hMSCs isolated from bone marrow were included in all studies as the control. Lastly, a mechanistic study was conducted to understand why hiMPCs and hMSCs behave differently in responding to TGFß. RESULTS: Chondrogenic medium supplemented with TGFß3 and BMP6 led to robust in vitro cartilage formation from hiMPCs with minimal hypertrophy. Cartilage tissue generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and resulted in a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFß3 induced phosphorylation of both Smad2/3 and Smad1/5 in hMSCs, but only activated Smad2/3 in hiMPCs. Supplementing BMP6 activated Smad1/5 and significantly enhanced TGFß's compacity in inducing hiMPC chondrogenesis. The chondro-promoting function of BMP6 was abolished by the treatment of a BMP pathway inhibitor. CONCLUSIONS: This study describes a robust method to generate chondrocytes from hiMPCs with low hypertrophy for hyaline cartilage repair, as well as elucidates the difference between hMSCs and hiMPCs in response to TGFß. Our results also indicated the importance of activating both Smad2/3 and Smad1/5 in the initiation of chondrogenesis.

Human Mesenchymal Stem Cell-Derived Miniature Joint System for Disease Modeling and Drug Testing.

Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell-derived microphysiological system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell-derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial-like fibrous tissue, and adipose tissue are integrated into a microfluidics-enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin-1ß mediated inflammation, which are similar to observations in joint diseases in humans. The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the "therapeutic effect" of the nonsteroidal anti-inflammatory drug, naproxen, as well as four other potential disease-modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions.

Engineering pre-vascularized bone-like tissue from human mesenchymal stem cells through simulating endochondral ossification.

Currently, most in vitro engineered bone tissues do not contain viable blood vessel systems, so the vascularization depends on post-implantation angiogenesis from the host, which is often insufficient for repairing large bone defects. In this study, we aimed to create pre-vascularized bone-like tissue from human bone marrow-derived mesenchymal stem cells (HBMSCs) within the self-generated extracellular matrix by simulating the developmental endochondral ossification. Afterward, a three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs)/HBMSCs was introduced to cover bone-like constructs surface for vascularization. Lastly, the pre-vascularized bone-like tissues were subcutaneously implanted into mice and the quality of newly formed blood vessels and bones were later assessed. We particularly examined whether the pre-existing HUVECs/HBMSCs vascular networks within the implants were able to integrate with the host's blood vessels and facilitate bone formation. Our results showed that this developmentally informed procedure resulted in a robust osteogenic differentiation of HBMSCs. Moreover, the bone-like constructs markedly promoted HUVEC/HBMSCs network formation in vitro. After 28 days of implantation in mice, the experimental group, in which bone-like constructs were pre-vascularized with HUVEC/HBMSCs networks, exhibited significantly more functional blood vessels than the control group that contained HUVEC and HBMSC single cells. Interestingly, increased levels of bone formation and absorption markers were also observed in the pre-vascularized bone-like constructs. Taken together, these findings demonstrated the potential of pre-vascularized bone-like constructs in repairing bone defects.

Engineering osteoarthritic cartilage model through differentiating senescent human mesenchymal stem cells for testing disease-modifying drugs.

Significant cellular senescence has been observed in cartilage harvested from patients with osteoarthritis (OA). In this study, we aim to develop a senescence-relevant OA-like cartilage model for developing disease-modifying OA drugs (DMOADs). Specifically, human bone marrow-derived mesenchymal stromal cells (MSCs) were expanded in vitro up to passage 10 (P10-MSCs). Following their senescent phenotype formation, P10-MSCs were subjected to pellet culture in chondrogenic medium. Results from qRT-PCR, histology, and immunostaining indicated that cartilage generated from P10-MSCs displayed both senescent and OA-like phenotypes without using other OA-inducing agents, when compared to that from normal passage 4 (P4)-MSCs. Interestingly, the same gene expression differences observed between P4-MSCs and P10-MSC-derived cartilage tissues were also observed between the preserved and damaged OA cartilage regions taken from human samples, as demonstrated by RNA Sequencing data and other analysis methods. Lastly, the utility of this senescence-initiated OA-like cartilage model in drug development was assessed by testing several potential DMOADs and senolytics. The results suggest that pre-existing cellular senescence can induce the generation of OA-like changes in cartilage. The P4- and P10-MSCs derived cartilage models also represent a novel platform for predicting the efficacy and toxicity of potential DMOADs on both preserved and damaged cartilage in humans.

Graphene oxide-functionalized nanocomposites promote osteogenesis of human mesenchymal stem cells via enhancement of BMP-SMAD1/5 signaling pathway.

Biomaterials that can harness the intrinsic osteogenic potential of stem cells offer a promising strategy to accelerate bone regeneration and repair. Previously, we had used methacrylated gelatin (GelMA)-based scaffolds to achieve bone formation from human mesenchymal stem cells (hMSCs). In this study, we aimed to further enhance hMSC osteogenesis by incorporating graphene oxide (GO)-based nanosheets into GelMA. In vitro results showed high viability and metabolic activities in hMSCs encapsulated in the newly developed nanocomposites. Incorporation of GO markedly increased mineralization within hMSC-laden constructs, which was further increased by replacing GO with silica-coated graphene oxide (SiGO). Mechanistic analysis revealed that the nanosheet enhanced the production, retention, and biological activity of endogenous bone morphogenetic proteins (BMPs), resulting in robust osteogenesis in the absence of exogenous osteoinductive growth factors. Specifically, the osteoinductive effect of the nanosheets was abolished by inhibiting the BMP signaling pathway with LDN-193189 treatment. The bone formation potential of the technology was further tested in vivo using a mouse subcutaneous implantation model, where hMSCs-laden GO/GelMA and SiGO/GelMA samples resulted in bone volumes 108 and 385 times larger, respectively, than the GelMA control group. Taken together, these results demonstrate the biological activity and mechanism of action of GO-based nanosheets in augmenting the osteogenic capability of hMSCs, and highlights the potential of leveraging nanomaterials such as GO and SiGO for bone tissue engineering applications.

Osteochondral Tissue Chip Derived From iPSCs: Modeling OA Pathologies and Testing Drugs.

Osteoarthritis (OA) is a chronic disease mainly characterized by degenerative changes in cartilage, but other joint elements such as bone are also affected. To date, there are no disease-modifying OA drugs (DMOADs), owing in part to a deficiency of current models in simulating OA pathologies and etiologies in humans. In this study, we aimed to develop microphysiological osteochondral (OC) tissue chips derived from human induced pluripotent stem cells (iPSCs) to model the pathologies of OA. We first induced iPSCs into mesenchymal progenitor cells (iMPCs) and optimized the chondro- and osteo-inductive conditions for iMPCs. Then iMPCs were encapsulated into photocrosslinked gelatin scaffolds and cultured within a dual-flow bioreactor, in which the top stream was chondrogenic medium and the bottom stream was osteogenic medium. After 28 days of differentiation, OC tissue chips were successfully generated and phenotypes were confirmed by real time RT-PCR and histology. To create an OA model, interleukin-1ß (IL-1ß) was used to challenge the cartilage component for 7 days. While under control conditions, the bone tissue promoted chondrogenesis and suppressed chondrocyte terminal differentiation of the overlying chondral tissue. Under conditions modeling OA, the bone tissue accelerated the degradation of chondral tissue which is likely via the production of catabolic and inflammatory cytokines. These findings suggest active functional crosstalk between the bone and cartilage tissue components in the OC tissue chip under both normal and pathologic conditions. Finally, a selective COX-2 inhibitor commonly prescribed drug for OA, Celecoxib, was shown to downregulate the expression of catabolic and proinflammatory cytokines in the OA model, demonstrating the utility of the OC tissue chip model for drug screening. In summary, the iPSC-derived OC tissue chip developed in this study represents a high-throughput platform applicable for modeling OA and for the screening and testing of candidate DMOADs.