Patterned, organoid-based cartilaginous implants exhibit zone specific functionality forming osteochondral-like tissues in vivo.

Tissue engineered constructs have the potential to respond to the unmet medical need of treating deep osteochondral defects. However, current tissue engineering strategies struggle in the attempt to create patterned constructs with biologically distinct functionality. In this work, a developmentally-inspired modular approach is proposed, whereby distinct cartilaginous organoids are used as living building blocks. First, a hierarchical construct was created, composed of three layers of cartilaginous tissue intermediates derived from human periosteum-derived cells: (i) early (SOX9), (ii) mature (COL2) and (iii) (pre)hypertrophic (IHH, COLX) phenotype. Subcutaneous implantation in nude mice generated a hybrid tissue containing one mineralized and one non-mineralized part. However, the non-mineralized part was represented by a collagen type I positive fibrocartilage-like tissue. To engineer a more stable articular cartilage part, iPSC-derived cartilage microtissues (SOX9, COL2; IHH neg) were generated. Subcutaneous implantation of assembled iPSC-derived cartilage microtissues resulted in a homogenous cartilaginous tissue positive for collagen type II but negative for osteocalcin. Finally, iPSC-derived cartilage microtissues in combination with the pre-hypertrophic cartilage organoids (IHH, COLX) could form dual tissues consisting of i) a cartilaginous safranin O positive and ii) a bony osteocalcin positive region upon subcutaneous implantation, corresponding to the pre-engineered zonal pattern. The assembly of functional building blocks, as presented in this work, opens possibilities for the production of complex tissue engineered implants by embedding zone-specific functionality through the use of pre-programmed living building blocks.

Human pluripotent stem cell-derived cartilaginous organoids promote scaffold-free healing of critical size long bone defects.

BACKGROUND: Bones have a remarkable capacity to heal upon fracture. Yet, in large defects or compromised conditions healing processes become impaired, resulting in delayed or non-union. Current therapeutic approaches often utilize autologous or allogeneic bone grafts for bone augmentation. However, limited availability of these tissues and lack of predictive biological response result in limitations for clinical demands. Tissue engineering using viable cell-based implants is a strategic approach to address these unmet medical needs. METHODS: Herein, the in vitro and in vivo cartilage and bone tissue formation potencies of human pluripotent stem cells were investigated. The induced pluripotent stem cells were specified towards the mesodermal lineage and differentiated towards chondrocytes, which subsequently self-assembled into cartilaginous organoids. The tissue formation capacity of these organoids was then challenged in an ectopic and orthotopic bone formation model. RESULTS: The derived chondrocytes expressed similar levels of collagen type II as primary human articular chondrocytes and produced stable cartilage when implanted ectopically in vivo. Upon targeted promotion towards hypertrophy and priming with a proinflammatory mediator, the organoids mediated successful bridging of critical size long bone defects in immunocompromised mice. CONCLUSIONS: These results highlight the promise of induced pluripotent stem cell technology for the creation of functional cartilage tissue intermediates that can be explored for novel bone healing strategies.

From skeletal development to the creation of pluripotent stem cell-derived bone-forming progenitors.

Bone has many functions. It is responsible for protecting the underlying soft organs, it allows locomotion, houses the bone marrow and stores minerals such as calcium and phosphate. Upon damage, bone tissue can efficiently repair itself. However, healing is hampered if the defect exceeds a critical size and/or is in compromised conditions. The isolation or generation of bone-forming progenitors has applicability to skeletal repair and may be used in tissue engineering approaches. Traditionally, bone engineering uses osteochondrogenic stem cells, which are combined with scaffold materials and growth factors. Despite promising preclinical data, limited translation towards the clinic has been observed to date. There may be several reasons for this including the lack of robust cell populations with favourable proliferative and differentiation capacities. However, perhaps the most pertinent reason is the failure to produce an implant that can replicate the developmental programme that is observed during skeletal repair. Pluripotent stem cells (PSCs) can potentially offer a solution for bone tissue engineering by providing unlimited cell sources at various stages of differentiation. In this review, we summarize key embryonic signalling pathways in bone formation coupled with PSC differentiation strategies for the derivation of bone-forming progenitors.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Folic Acid Exposure Rescues Spina Bifida Aperta Phenotypes in Human Induced Pluripotent Stem Cell Model.

Neural tube defects (NTDs) are severe congenital abnormalities, caused by failed closure of neural tube during early embryonic development. Periconceptional folic acid (FA) supplementation greatly reduces the risk of NTDs. However, the molecular mechanisms behind NTDs and the preventive role of FA remain unclear. Here, we use human induced pluripotent stem cells (iPSCs) derived from fetuses with spina bifida aperta (SBA) to study the pathophysiology of NTDs and explore the effects of FA exposure. We report that FA exposure in SBA model is necessary for the proper formation and maturation of neural tube structures and robust differentiation of mesodermal derivatives. Additionally, we show that the folate antagonist methotrexate dramatically affects the formation of neural tube structures and FA partially reverts this aberrant phenotype. In conclusion, we present a novel model for human NTDs and provide evidence that it is a powerful tool to investigate the molecular mechanisms underlying NTDs, test drugs for therapeutic approaches.

Combinatorial Analysis of Growth Factors Reveals the Contribution of Bone Morphogenetic Proteins to Chondrogenic Differentiation of Human Periosteal Cells.

Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-ß1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (µMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p < 0.001), which in combination with BMP6 synergistically enhanced cartilage-like tissue formation that displayed in vitro mineralization capacity at day 14 (p < 0.001). Gene expression of µMasses cultured for 7 days with a medium formulation supplemented with 100 ng/mL of BMP2 and BMP6 and a low concentration of GDF5, TGF-ß1, and FGF2 showed increased expression of Sox9 (1.7-fold) and the matrix molecules aggrecan (7-fold increase) and COL2A1 (40-fold increase) compared to nonstimulated control µMasses. The DoE analysis indicated that in GF combinations, BMP2 was the strongest effector for chondrogenic differentiation of hPDCs. When transplanted ectopically in nude mice, the in vitro-differentiated µMasses showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This study indicates the power of using the DoE approach for the creation of new medium formulations for skeletal tissue engineering approaches.

Sox9 reprogrammed dermal fibroblasts undergo hypertrophic differentiation in vitro and trigger endochondral ossification in vivo.

Strategies for bone regeneration are undergoing a paradigm shift, moving away from the replication of end-stage bone tissue and instead aiming to recapture the initial events of fracture repair. Although this is known to resemble endochondral bone formation, chondrogenic cell types with favorable proliferative and hypertrophic differentiation properties are lacking. Recent advances in cellular reprogramming have allowed the creation of alternative cell populations with specific properties through the forced expression of transcription factors. Herein, we investigated the in vitro hypertrophic differentiation and in vivo tissue formation capacity of induced chondrogenic cells (iChon cells) obtained through direct reprogramming. In vitro hypertrophic differentiation was detected in iChon cells that contained a doxycycline-inducible expression system for Klf4, cMyc, and Sox9. Furthermore, endochondral bone formation was detected after implantation in nude mice. The bone tissue was derived entirely from host origin, whereas cartilage tissue contained cells from both host and donor. The results obtained highlight the promise of cellular reprogramming for the creation of functional skeletal cells that can be used for novel bone healing strategies.

Humanized culture of periosteal progenitors in allogeneic serum enhances osteogenic differentiation and in vivo bone formation.

The translation of stem cell-based regenerative solutions from the laboratory to the clinic is often hindered by the culture conditions used to expand cell populations. Although fetal bovine serum (FBS) is widely used, regulatory bodies and safety concerns encourage alternative, xeno-free culturing practices. In an attempt to apply this approach to a bone-forming combination product of human periosteal progenitors (human periosteum derived cells) on a clinically used calcium phosphate carrier, FBS was substituted for human allogeneic serum (hAS) during cell expansion. It was found that cell proliferation was increased in hAS along with an apparent commitment to the osteogenic lineage, indicated by enhanced Runx2 expression, as well as alkaline phosphatase activity and matrix mineralization. Following analysis of signaling pathways, it was found that interferon-mediated signaling was downregulated, whereas JAK-STAT signaling was upregulated. STAT3 phosphorylation was enhanced in hAS-cultured human periosteum derived cells, inhibition of which ablated the proliferative effect of hAS. Furthermore, following in vivo implantation of hAS-cultured cells on NuOss scaffolds, enhanced bone formation was observed compared with FBS (71% increase, p < .001). Interestingly, the de novo-formed bone appeared to have a higher ratio of immature regions to mature regions, indicating that after 8 weeks implantation, tissue-formation processes were continuing. Integration of the implant with the environment appeared to be altered, with a decrease in calcium phosphate grain size and surface area, indicative of accelerated resorption. This study highlights the advantages of using humanized culture conditions for the expansion of human periosteal progenitors intended for bone regeneration.