Directing iPSC differentiation into iTenocytes using combined scleraxis overexpression and cyclic loading

Abstract

Regenerative therapies for tendon are falling behind other tissues due to the lack of an appropriate and potent cell therapeutic candidate. This study aimed to induce tenogenesis using stable Scleraxis (Scx) overexpression in combination with uniaxial mechanical stretch of iPSC‐derived mesenchymal stromal‐like cells (iMSCs). Scx is the single direct molecular regulator of tendon differentiation known to date. Bone marrow–derived (BM‐)MSCs were used as reference. Scx overexpression alone resulted in significantly higher upregulation of tenogenic markers in iMSCs compared to BM‐MSCs. Mechanoregulation is known to be a central element guiding tendon development and healing. Mechanical stimulation combined with Scx overexpression resulted in morphometric and cytoskeleton‐related changes, upregulation of early and late tendon markers, and increased extracellular matrix deposition and alignment, and tenomodulin perinuclear localization in iMSCs. Our findings suggest that these cells can be differentiated into tenocytes and might be a better candidate for tendon cell therapy applications than BM‐MSCs.

iPSC‐derived tenocytes seeded on microgrooved 3D printed scaffolds for Achilles tendon regeneration

“…Furthermore, studies of tendon development using SCX −/ − mice indicate SCX is responsible for activating later tendon differentiation markers including Collagen type 1A1 and Tenomodulin 22,23 . Our group has previously shown the tenogenic differentiation potential of iMSCs, stimulated by SCX overexpression and biomechanical tension in vitro 18 …”

“…17 The iMSCs were then transduced with a SCX-GFP plasmid (OriGene) using a lentiviral vector as previously published by our group. 18 2.2 | 3D printing of PCL scaffolds 3D printed constructs were fabricated by melt extrusion using a 3D Discovery printer (RegenHu Ltd.). Rectangular constructs (filaments oriented at angles of ±30°, 1.25 mm spacing between filaments, 0.4 mm layer height) were designed using BioCAD software (RegenHu Ltd., version 1.1-12), and were printed with 2 or 4 layers having overall dimensions of 2.5 × 5 mm or 5 × 10 mm for small and large sizes, respectively (Figure 1A).…”

“…15,16 In our previous work we utilized iPSC-derived MSCs (iMSCs) for musculoskeletal tissue repair. [17][18][19] Scleraxis (SCX) is a transcription factor that has been identified as one of the earliest detectable markers for tendon differentiation and is necessary for the proper embryonic development of tendons. 20,21 Current studies show that, during tendon development, SCX is the only transcription factor necessary for tendon differentiation.…”

“…22,23 Our group has previously shown the tenogenic differentiation potential of iMSCs, stimulated by SCX overexpression and biomechanical tension in vitro. 18 Scaffolds have become an integral component of tendon tissue engineering strategies and are usually designed to provide cells with a fibrous network mimicking the native tendon microenvironment while minimizing cell migration to other areas of the body and providing biomechanical support. These architectural features are most commonly achieved with electrospinning of synthetic thermoplastic polymers, such as polycaprolactone (PCL), polyglycolic acid (PGA), and poly-dl-lactic-co-glycolic acid (PLGA), due to their degradability, capability to allow cell attachment, and supportive mechanical properties.…”

“…Small animal tendon repair models utilizing iPSCs demonstrated improved functional outcomes 15,16 . In our previous work we utilized iPSC‐derived MSCs (iMSCs) for musculoskeletal tissue repair 17–19 …”