Authors:

April Craft

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37% of adults have a degenerative cartilage disease. Today, patients are offered Autologous Chondrocyte Implantation, wherein cartilage is replaced with patient cells, however this cartilage does not contain the gradient of layers in native cartilage and often fails to integrate into host tissue. Stem cell-based cartilage offers a promising alternative. In development, mesenchymal stem cells become chondrocytes and transdifferentiate into bone through endochondral ossification. Through directed differentiation, induced pluripotent stem cells (iPSCs) may develop into chondrocytes and then transdifferentiate into bone, generating a tissue containing bone and cartilage. We aim to optimize this process by determining factors required to transition chondrocytes into osteoblasts. We hypothesized that by enhancing hypertrophy, supplementing with phosphate, and activating Wnt signalling, iPSC-derived hypertrophic chondrocytes will develop a robust and mineralized Type X Collagen matrix, and hypertrophic chondrocytes will transdifferentiate into osteoblasts. Human iPSCs were differentiated into hypertrophic chondrocytes via TGF-β treatment (2 weeks) followed by BMP-4 treatment (up to 12 weeks). Enhanced hypertrophy was induced using an Indian Hedgehog Pathway activator or thyroid hormone. Phosphate was added to tissues to induce mineralization of the Type X Collagen matrix. Wnt activators were added to induce chondrocyte to osteoblast transdifferentiation. Hypertrophy and mineralization was quantified using RT-qPCR for COL10 expression and visualized through histological analysis for Type X Collagen, glycosaminoglycan, and alkaline phosphatase (ALP). Preliminary results indicate Indian Hedgehog Pathway activation increased the number of hypertrophic chondrocytes but degraded tissue integrity, reducing glycosaminoglycans. Thyroid hormone enhanced hypertrophy by increasing hypertrophic chondrocytes and increasing glycosaminoglycan production. Phosphate supplementation mineralized the tissue matrix after eight weeks of BMP-4 treatment, as indicated by ALP staining. RT- qPCR and Wnt signaling results are forthcoming.This study provides preliminary insights into an iPSC-derived endochondral ossification model that may generate cartilage-bone tissue constructs for treating degenerative cartilage diseases. Closed Loop Automation For Engineering Orthogonal Translation Systems Nayan Sapers, Felix Radford, George Church Harvard | Molecular and Cellular Biology | 2026 Orthogonal translation systems (OTSs) enable incorporation of nonstandard amino acids (nsAAs) into proteins, expanding their functional capabilities. Traditional OTS development relies on directed evolution with limited throughput, constraining exploration of design space. This project develops closed-loop automation to engineer chimeric aminoacyl-tRNA synthetases with unprecedented orthogonality and efficiency. The approach integrates three aims: complete automation of tRNA- SCAN on Opentrons Flex, automated DNA library assembly, and AI-guided design of novel OTSs. tRNA-SCAN is a procedure developed in lab that enables massively parallel profiling of aminoacylation specificity by converting tRNA charging states into sequenceable outputs through enzymatic modifications. The automated workflow encompasses periodate oxidation, dephosphorylation, adapter ligations, reverse transcription, and library preparation. Current progress demonstrates successful automation of RNA production and purification with yields comparable to manual methods. The DNA assembly pipeline combines PCR amplification, magnetic bead purification, and Gibson Assembly to construct chimeric synthetase libraries. This automation enables construction of thousands of variants weekly, compared to approximately 100 manually. Future work integrates ESM-3 and AlphaFold3 for computational design of linker sequences and binding domains. The closed- loop optimization strategy combines high-throughput experimental characterization with machine learning to iteratively improve designs. This integrated approach promises to overcome current limitations in OTS development, enabling reliable multi-site nsAA incorporation for advanced biotechnology applications. Harvard Summer Undergraduate Research Village Program for Research in Science and Engineering

Source:

Harvard / Human Developmental and Regenerative Biology / 2026

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Co-authors:

April Craft