mechanical loading of imscscx+ in a 2d bioreactor for cyclic stretching

Harnessing iPSC-Derived iTenocytes for Effective Cell-Based Therapy

To tackle the contemporary issues in tendon and ligament repair, there&#39;s a requirement for a pertinent cell candidate suitable for cell-based therapies. One avenue of investigation in tissue engineering for tendon repair involves the exploration of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and adipose tissue-derived stromal cells (ASCs) as potential strategies. These cells have multipotent capability, great abundance, and regenerative potential in vivo. Additionally, they have shown enhanced healing capacity and improved functional outcomes in animal models1. Nonetheless, these cells exhibit restricted self-renewal capabilities, phenotypic diversity, and notably, limited capacity for tendon formation. Induced pluripotent stem cell (iPSC) technology offers a solution to these constraints due to its remarkable self-renewal capacity and unmatched developmental adaptability. Our research team and others have achieved successful differentiation of iPSCs into mesenchymal stromal cell-like entities (iMSCs)2,3. As such, iMSCs have the potential to be an allogenic source for tendon cell therapy applications.
Scleraxis (SCX) is a transcription factor essential for tendon development and is considered the earliest detectable marker for differentiated tenocytes. Additionally, SCX activates downstream tendon differentiation markers, including type 1a1 chain collagen 1 (COL1a1), mohawk (MKX), and tenomodulin (TNMD), among others4,5,6. Other genes expressed during tendon maturation include tubulin polymerization-promoting protein family member 3 (TPPP3) and platelet-derived growth factor receptor alpha (PDGFRa)7. While these genes are essential for tendon development and maturation, they are unfortunately not unique to tendon tissue and are expressed in other musculoskeletal tissues like bone or cartilage5,7.
In addition to the expression of markers during tendon development, mechanostimulation is an essential element for embryonic tendon development and healing4,5,6. Tendons are mechanoresponsive, and their growth patterns change in response to their environment. At the molecular level, biomechanical cues affect the development, maturation, maintenance, and healing responses of tenocytes8. Various bioreactor systems have been utilized to model physiological loads and biomechanical cues. Some of these model systems include ex vivo tissue loading, 2D cell loading systems applying bi-axial or uniaxial tension, and 3D systems using scaffolds and hydrogels9,10. 2D systems are advantageous when studying the mechanical stimulation&#39;s effects on either tendon-specific genes or the morphology of the cells in the context of cell fate, while 3D systems can more accurately replicate cell-ECM interactions9,10.
In 2D loading systems, the strain between the cells and the culture substrate is homogeneous, meaning that the applied load on the cytoskeleton of the cells can be fully controlled. In comparison to bi-axial loading, uniaxial loading is more physiologically relevant, as tenocytes are predominantly subjected to uniaxial loading from collagen bundles in vivo9. It is found that during daily activities, tendons are subjected to uniaxial tensile loading up to 6% strain11. Specifically, previous studies have found that loading within the physiological ranges of 4%-5% has been shown to promote tenogenic differentiation by preserving tendon-related marker expression like SCX and TNMD, as well as increased collagen production9,10. Strains of more than 10% may be traumatically relevant but not physiologically relevant12,13.
Here, a protocol is presented that takes into account the synergistic effect of mechanical and biological stimulation that may be essential for the generation of tenocytes. We first describe a reproducible method to induce iPSCs into iMSCs via short-term exposure of embryoid bodies to growth factors, confirmed by MSC surface markers using flow cytometry. We then detail a lentiviral transduction method to engineer iMSCs to have stable overexpression of SCX (iMSCSCX+). For further cell maturation, the iMSCSCX+ are seeded into fibronectin-coated silicone plates and undergo an optimized uniaxial tension protocol using CellScale MCFX bioreactor. The tenogenic potential was confirmed by observing the upregulation of early and late tendon markers, as well as collagen deposition14. This method of generating iTenocytes is a proof-of-concept that may offer an unlimited off-the-shelf, allogeneic source for tendon cell therapy applications.

Author Spotlight: Advancements in Cell and Tissue Engineering for Tendon Repair 

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension

Tendon injury is one of the most prevalent musculoskeletal problems in the US, affecting an estimated 33 million people annually. Our research focuses on developing a cell and tissue engineering approach for repairing tendons that can drastically improve patient outcomes. Current technologies used to advance tendon research include the use of stem cells from different sources like induced pluripotent stem cells in combination with the use of bioreactors, genetic engineering biomaterials, and other tissue engineering techniques to mimic the environmental cues needed for tendon.Our understanding of how tendons develop from their origins is limited. There are several well-known genes that are very highly expressed during tendon maturation. However, they're unfortunately not unique to the tendon tissue.This makes it difficult for researchers to standardly assess successful tendon differentiation. A protocol takes advantage of remarkable IPC differentiation potential and what we know so far on tendon development. Our goal is to develop an allogeneic cell source for tendon cell therapy applications.Our laboratories focused on elucidating the pathways involved with tendon sulfate to better understand tendon development. We want to implement this knowledge in combination with tissue engineering technologies and biomaterials to develop more effective alternatives for traditional tendon repair.

Watch this Scientific Journal Video about Mechanical Loading of iMSCSCX+ in a 2D Bioreactor for Cyclic Stretching at JoVE.com

Mechanical Loading of iMSCSCX+ in a 2D Bioreactor for Cyclic Stretching  

Mechanical Loading of iMSCSCX+ in a 2D Bioreactor for Cyclic Stretching

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Research

JoVE Journal

Bioengineering

301 Views.  Cedars-Sinai Medical Center. Mechanical Loading of iMSCSCX+ in a 2D Bioreactor for Cyclic Stretching

Video: Mechanical Loading of iMSCSCX+ in a 2D Bioreactor for Cyclic Stretching  

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to promote tendon regeneration. Mesenchymal stromal cells (MSCs) have been explored as a potential tissue engineering strategy for tendon repair. While they are multipotent and have regenerative potential in vivo, they are limited in their self-renewal capacity and exhibit phenotypic heterogeneity. Induced pluripotent stem cells (iPSCs) can circumvent these limitations due to their high self-renewal capacity and unparalleled developmental plasticity. In tenocyte development, Scleraxis (Scx) is a crucial direct molecular regulator of tendon differentiation. Additionally, mechanoregulation has been shown to be a central element guiding embryonic tendon development and healing. As such, we have developed a protocol to encapsulate the synergistic effect of biological and mechanical stimulation that may be essential for generating tenocytes. iPSCs were induced to become mesenchymal stromal cells (iMSCs) and were characterized with classic mesenchymal stromal cell markers via flow cytometry. Next, using a lentiviral vector, the iMSCs were transduced to stably overexpress SCX (iMSCSCX+). These iMSCSCX+ cells can be further matured into iTenocytes via uniaxial tensile loading using a 2D bioreactor. The resulting cells were characterized by observing the upregulation of early and late tendon markers, as well as collagen deposition. This method of generating iTenocytes can be used to assist researchers in developing a potentially unlimited off-the-shelf allogeneic cell source for tendon cell therapy applications.

This article describes a procedure to produce iTenocytes by generating iPSC-derived mesenchymal stromal cells with combined overexpression of Scleraxis using a lentiviral vector and uniaxial stretching via a 2D bioreactor.

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to ...