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

Summary

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.

Abstract

Today'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 (iMSC^SCX+). These iMSC^SCX+ 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.

Introduction

To tackle the contemporary issues in tendon and ligament repair, there'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 others^4,5,6. 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 cartilage^5,7.

In addition to the expression of markers during tendon development, mechanostimulation is an essential element for embryonic tendon development and healing^4,5,6. 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 hydrogels^9,10. 2D systems are advantageous when studying the mechanical stimulation'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 interactions^9,10.

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 production^9,10. Strains of more than 10% may be traumatically relevant but not physiologically relevant^12,13.

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 (iMSC^SCX+). For further cell maturation, the iMSC^SCX+ 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.

Protocol

This protocol to produce iTenocytes can be conducted in three major steps: iPSCs to iMSCs (10 days), iMSC to iMSC^SCX+ (2 weeks), iMSC^SCX+ to iTenocytes (minimum 4 days). Each major step in the protocol can be paused and restarted later, depending on the experimental timeline. For methods involved with culturing of cells, sterile techniques should be employed. All cells in this protocol should be grown at 37 °C, 5% CO₂, and 95% humidity.

1. Human iPSC induction into induced Mesenchymal Stromal Cells (iMSCs)

1. Experiment preparation
   1. Prepare Iscove's Modified Dulbecco's Medium - Embryoid Bodies (IMDM-EB) media.
      1. Supplement 50 mL of IMDM basal media with 8.5 mL of knock-out serum replacement, 500 µL of Minimal Essential Medium (MEM) non-essential amino acids, 0.385 µL (110 mM stock) beta-mercaptoethanol, and 500 µL antibiotic-antimycotic solution (AAS) (final concentrations: 17%, 1%, 110 µM, 1%, respectively) (see Table of Materials).
   2. Prepare Mesenchymal Stromal Cell (MSC) media.
      1. Supplement 440 mL of low glucose Dulbecco's Modified Eagle Medium (DMEM) with 50 mL of fetal bovine serum (FBS), 5 mL of antibiotic-antimycotic solution (AAS), and 5 mL of L-glutamine (final concentrations: 10%, 1%, 2 mM, respectively).
   3. Prepare poly(2-hydroxyethyl methacrylate) (poly-HEMA) coated plates.
      1. Add 10 g of the poly-HEMA (see Table of Materials) to a sterile bottle.
      2. Add a magnetic stirrer to the bottle, and while stirring, slowly add 500 mL 95% EtOH.
      3. Once all the EtOH has been added, turn the stirring mode on at 1200 rpm with additional low heat for at least 6 h. The poly-HEMA solution can be stored at room temperature for up to a year.
      4. Under sterile conditions, add 2.5 µg/cm² in 9 mL to a T75 flask. Adjust the volume according to the size of the culture vessel.
      5. Maintaining sterility, allow the vessels to air dry for the EtOH to completely evaporate before use. This usually takes 24-72 h.
   4. Prepare gelatin-coated flasks.
      1. Wash a glass bottle with NaOH and dedicate it to the gelatin preparation. Do not use detergent.
      2. Prepare 1% gelatin solution (5 g gelatin and 500 mL of endotoxin-free water). Autoclave the glass bottle with the gelatin solution for 30 min.
      3. Allow the gelatin solution to cool, and it can be stored at room temperature.
      4. Coat one T75 flask with 5 mL of gelatin solution per flask and incubate for at least 1 h prior to seeding cells. Gelatin-coated flasks can be prepared 1 day in advance.
   5. Prepare fluorescence-activated cell sorting (FACS) buffer.
      1. Mix PBS with 2% bovine serum albumin and 0.1% sodium azide. Store at 4 °C.
2. Generation of embryoid bodies (EBs)
   NOTE: iPSCs should be cultured in a 6-well plate and should reach 70%-80% confluence before use.
   1. On day 0, bring a 384 clear-bottomed PCR plate in the tissue culture hood and UV for 15 min without a lid.
   2. Remove the growth medium from the iPSCs (see Table of Materials) and wash the wells with PBS.
   3. Add 0.5 mL of pre-warmed gentle cell dissociation reagent (see Table of Materials) to each well and incubate for 5 min at 37 °C. Observe the cells every 5 min until the iPSCs have become single cells or small aggregates lifted in suspension.
   4. Resuspended the cell suspension with a 1 mL pipette to ensure a single cell suspension.
   5. Centrifuge the cells at 300 x g for 5 min at room temperature.
   6. Remove the supernatant, resuspend in IMDM-EB media (step 1.1.1), and count the viable cells using a hemocytometer or a cell counter.
   7. Calculate the appropriate volume of cell suspension required to achieve 5,000-25,000 cells per well of the 384-well plate with 25 µL of cell suspension per well.
      NOTE: Generally, 2-3 confluent (70%-80%) wells of a 6-well-plate can make EBs in one 384-well plate. The size of cells per EB will be determined empirically. For an entire 384-well plate, 9.6 mL of cell suspension must be used, 25 µL per well.
   8. Centrifuge the cells down again at 300 x g for 5 min at room temperature.
   9. Remove the supernatant and resuspend the cells in an appropriate volume of IMDM-EB media and 10 µM of Y-27632 dihydrochloride (stock: 10 mM, 1000x, see Table of Materials) in a pre-chilled 15 mL conical tube.
   10. Add cold solubilized basement membrane matrix (1 mg per 384-well plate worth of EBs) to the conical (see Table of Materials).
   11. Distribute 25 µL of the cell suspension to each well. Place a sterile lid on the plate and spin at 450 x g at 4 °C for 7 min. Incubate at 37 °C for 48 h.
   12. On day 2, transfer the cells to a 100 mm plate with 3 mL of pre-warmed IMDM-EB media.
   13. Transfer the EBs to the dedicated poly-HEMA-coated T75 flasks with 10 mL of IMDM-EB. Adjust the volume according to the size of the culture vessel. Allow the EBs to grow for 3 days.
   14. On day 5, transfer the EBs to the prepared gelatin-coated T75 flasks with 10 mL of IMDM-EB media. Adjust the volume according to the size of the culture vessel. Grow the EBs for 3 more days in suspension.
   15. On day 8, ensure that two types of EBs are observed: EBs that are attached to the flask, and non-attached EBs. Wash out the non-attached EBs from the flask with PBS.
   16. To the attached EBs, add IMDM-EB media supplemented with TGFβ-1 (10 ng/mL) (see Table of Materials) and allow them to grow for 2 days.
   17. On day 10, change the medium to MSC medium. Cells should be fed twice a week. Once 70% confluent, proceed to the "Standard lifting cells protocol" to replate cells. Replate the cells on gelatin-coated plates.
3. Standard lifting cells protocol
   1. Once the adherent cells have reached 70% confluence, aspirate the growth medium from the plates and wash with PBS.
   2. Lift the cells by adding 5 mL of pre-warmed 0.25% trypsin to each 150 mm plate and incubate for 5 min at 37 °C. Adjust trypsin volume according to the size of the culture vessel.
   3. Under a microscope, check that most of the cells have been lifted from each plate. If there are still cells attached, gently tap the sides of the plates to dislodge the cells.
   4. Collect the cells into a conical tube by adding double the volume of the pre-warmed growth medium.
   5. Centrifuge the cells at room temperature for 5 min at 300 x g. Remove the supernatant and proceed to the next steps.
4. Flow cytometry assessment for MSC marker expression
   ​NOTE: Human MSCs, like bone marrow MSCs, should be used as the positive control.
   1. Perform the "Standard lifting cells protocol" (step 1.3).
   2. Resuspend the supernatant with 3 mL of FACS buffer and transfer the whole volume to FACS tubes.
   3. Centrifuge at room temperature for 5 min at 300 x g. Repeat the wash step with FACS buffer two more times.
   4. To stained tubes, add 2 µL of mouse antihuman CD90-FITC, mouse antihuman CD44-APC, and antihuman CD105-PE (see Table of Materials) and incubate for 45 min at 4 °C.
   5. To the isotype control tubes, add 20 µL of mouse IgG2a-FITC, mouse IgG1-PB, and mouse IgG1-PE (see Table of Materials).
   6. Incubate in the dark at 4 °C for 15 min. Wash all the tubes by adding 3 mL of FACS buffer and centrifuging for 5 min at 300 x g (at room temperature). Resuspend the cells in 250 µL of FACS buffer for each tube.
   7. Analyze the expression of the MSCs surface markers using flow cytometry¹⁴. Excitation and emission wavelengths should be: CD90-FITC, excitation peak at 495 nm and emission peak at 519 nm; CD44-APC, excitation peak at 640 and emission peak at 660 nm; CD105-PE, excitation peak at 561 nm and emission peak at 574 nm.

2. iMSC passaging and expansion

1. Preparing reagents
   1. Prepare MSC freeze media.
      1. Combine 30 mL of low glucose DMEM, 5 mL of DMSO, and 15 mL of FBS (final concentrations: 60%, 10%, 30%, respectively.)
      2. Filter the solution using a sterile 0.45 µm filter. Store at 4 °C.
2. Passaging
   NOTE: Following induction, iMSCs must be grown on gelatin-coated plates for an additional 2 passages before being weaned off to growing on plastic tissue culture plates. Additionally, cells should be passaged with a 1:3 split ratio when 70% confluent.
   1. Perform the "Standard lifting cells protocol" (step 1.3).
   2. Resuspend the cells in MSC media, and equally distribute the cells into three new 150 mm plates of T175 flasks with 20 mL of media per flask. Return the cells to 37 °C.
   3. Feed the cells twice a week by replacing the growth medium with pre-warmed MSC media.
3. Freezing
   1. Perform the "Standard lifting cells protocol".
   2. Resuspend the cells in 1 mL of MSC freeze media. Add the 1 mL of cell suspension to a cryovial and store in a freezing container for 24 h at -80 °C before transferring to liquid nitrogen for long-term storage.
4. Thawing
   1. Prepare 10 mL of pre-warmed MSC media in a 15 mL conical tube.
   2. Retrieve the cryovial, dip it into a 37 °C water bath, and swirl until a pea-sized ice ball remains (approximately 1-2 min).
   3. In the cell culture hood, slowly add 1 mL of the pre-warmed media to the cryovial and pipette up and down to mix.
   4. Transfer the cell suspension from the cryovial to the prepared 15 mL conical tube with pre-warmed media, and centrifuge for 5 min at 300 x g (at room temperature).
   5. Remove the supernatant and resuspend with fresh media. Add the cell suspension to a 150 mm plate with a total volume of 20 mL. Adjust the volume according to the size of the culture flask.
   6. Incubate the cells at 37 °C until ready to split.

3. Genetic engineering of iMSCs to overexpress SCX using lentiviral transduction

NOTE: This section of the protocol takes two weeks to complete.

1. Preparation of media and reagents
   1. Prepare HEK293T/17 cell media.
      1. Supplement 445 mL of Eagle's Minimum Essential Medium (EMEM) with 50 mL of FBS and 5 mL of AAS (final concentrations: 10%, 1%, respectively).
   2. Prepare MSC lenti-packing media.
      1. Prepare the heat-inactivated serum by heating 50 mL of room temperature FBS in a 60 °C water bath for 30 min.
      2. Combine 445 mL of low glucose DMEM with the heat-inactivated FBS and 5 mL of L-glutamine (final concentrations: 10%, 2 mM, respectively.)
   3. Prepare transfection complex.
      1. Allow transfection complex components (see Table of Materials) to thaw on ice: packaging plasmids VSV-G and Delta, SCX plasmid, and BioT^15,16.
      2. Gently vortex and spin down the plasmids. Measure the DNA concentrations.
      3. Based on the DNA concentrations and the number of flasks intended for transfection, calculate the volume of each component needed: for one T75 flask, the transfection complex will consist of 750 µL serum-free medium (like serum-free DMEM or PBS), 7.5 µg SCX plasmid, 0.75 µg VSV-G plasmid, 6.75 µg Delta plasmid, and 22.5 µL BioT. Consider extra for pipetting error.
      4. Mix by gently pipetting up and down. Spin briefly in a centrifuge. Incubate at room temperature for 15 min and use immediately.
2. Transfection and lentiviral production
   CAUTION: Beginning from day 3, the protocol entails working with lentivirus, and thus, the work must be carried out using containment level 2+ operational procedures. Workers must wear personal protective equipment, including two layers of gloves, wrist coverings, safety glasses, a mask, and long pants and closed shoes. All waste and materials, including pipette tips, flasks, and liquid medium, must be decontaminated with bleach (final 1% sodium hypochlorite) for at least 20 min. Do not use the vacuum.
   1. Seed HEK293T/17 cells.
      1. Approximately 18-24 h prior to transfection (day 0), lift and plate the 293T cells into T75 flasks. The following volumes assume T75 as the culture vessel. Adjust the volume according to the cell culture vessel used.
      2. Remove the culture medium from the flasks and wash with 5 mL of PBS.
         NOTE: The cells very easily lift from the surface of the flask. Add PBS to the side of the flask and avoid direct addition of solution to the cells.
      3. Add 3 mL of pre-warmed 0.25% trypsin to each flask and incubate at 37 °C for 5 min. Collect the cells into a conical tube and centrifuge at room temperature for 5 min at 300 x g.
      4. Remove the supernatant, resuspend in 293T media, and count the viable cells using a hemocytometer or a cell counter.
      5. Plate the cells at 5.3 x 10⁴ cells/cm² and incubate at 37 °C overnight.
      6. The next day (day 1), prepare the transfection complex. Replace the growth medium from the cells with 5 mL of pre-warmed MSC lenti-packaging medium (step 3.1.2).
      7. Add the transfection complex dropwise to the cells. Gently tilt the dish to ensure even exposure of the transfection complex to the cells. Return the cells to the incubator for 48 h.
         NOTE: Toxicity should be observed after 24 h (day 2).
      8. After 48 h (day 3), carefully collect the virus-containing medium into a separate conical tube using a pipette, and centrifuge for 5 min at 300 x g (at room temperature).
      9. Add 5 mL of pre-warmed MSC lenti-packaging medium to the T75 flask of 293T cells and return to the incubator overnight.
      10. After centrifugation, filter the supernatant with a 0.45 µm sterile filter by directly pipetting the supernatant through the filter. Store the virus-containing medium at 4 °C overnight.
      11. After another 24 h (day 4), once again carefully collect the virus-containing medium into a separate conical tube using a pipette, and centrifuge for 5 min at 300 x g (at room temperature).
      12. Combine the virus with the previous collection day virus and mix to ensure a homogenous solution. At this point, the protocol can be paused and restarted later, depending on the experimental timeline. The lentivirus can be aliquoted and frozen at -80 °C, or the lentivirus can be stored on ice while proceeding with "Transduction of iMSCs with SCX" (step 3.3).
          NOTE: Once the lentivirus aliquots are frozen and thawed, do not refreeze.
3. Transduction of iMSCs with SCX
   1. Perform titer plate transduction.
      1. On day 0, perform the "Standard lifting cells protocol".
      2. Resuspend the cells in MSC media and count the viable cells using a hemocytometer or a cell counter.
      3. In a 6-well plate, add 4 x 10³ cells/cm². Replate the remaining in an extra 150 mm plate with 20 mL of MSC media (this will be the control plate). Incubate the cells for 24 h at 37 °C.
      4. The next day (day 1), the cells should be ~40% confluent. Prewarm the lenti-packaging growth medium and thaw the polybrene and approximately 3 mL of lentivirus on ice (or if done on the same day as virus collection, store on ice until use).
      5. Add lenti-packaging growth medium and lentivirus to the wells based on the desired titers (i.e., 12.5%, 25%, 50%, 75%, 100%). Add 0.5 µL of polybrene to achieve a final concentration of 5 µg/mL (stock concentration: 10 mg/mL, see Table of Materials).
      6. Swirl the plate around to ensure even exposure to all the cells. Return the plate to the incubator at 37 °C for 48 h.
   2. Assess the SCX lentivirus titer efficiency with flow cytometry.
      1. After 48 h, perform the "Standard lifting cells protocol".
      2. Resuspend the cells in PBS and count viable cells using a hemocytometer or cell counter. Centrifuge the cells at room temperature for 5 min at 300 x g.
      3. Remove the supernatant and resuspend it in 3 mL of FACS buffer to wash. Transfer the cell suspension to flow cytometry tubes and centrifuge for 5 min at 300 x g.
      4. Repeat the FACS buffer wash two more times and resuspend the final cell pellets in 300 µL of FACS buffer. Assess the expression of GFP for each titer using a flow cytometry machine.
      5. Based on the titers and cell viability counts, determine the appropriate lentivirus titer to proceed with the transduction. At this point, the protocol can be paused and restarted later, depending on the experimental timeline.
   3. Perform large scale SCX transduction.
      NOTE: The listed volumes are per 1x 150 mm plate or 1x T175 flask. This may be adjusted accordingly depending on desired vessel size and scale of transduction.
      1. On day 0, perform the "Standard lifting cells protocol".
      2. Resuspend the cells in MSC media and count the viable cells using a hemocytometer or a cell counter. Seed the cells at 4 x 10³ cells/cm². Incubate the cells for 24 h at 37 °C.
      3. The next day (day 1), the cells should be ~40% confluent. Prewarm the lenti-packaging growth medium and thaw the polybrene and precalculated amount of lentivirus on ice.
      4. Based on the decided titer, add the desired amount of lenti-packaging growth medium and lentivirus to the wells. Add 5 µg/mL of polybrene (stock concentration: 10 mg/mL).
      5. On day 3, observe the cells under a fluorescent microscope. The newly produced iMSC^SCX+ should fluoresce GFP. Replace the virus-containing medium with pre-warmed MSC media. At this point, the protocol can be paused and restarted later, depending on the experimental timeline.

4. iMSC^SCX+ passaging and expansion

1. Perform passaging, expanding, freezing, and thawing of iMSC^SCX+ following the same as for iMSCs, as described in step 2 of the protocol.

5. Mechanical loading

NOTE: This section takes a minimum of 4 days but can be longer depending on whether cell contraction is observed.

1. Experiment preparation
   1. Prepare the silicone plates.
      1. Autoclave 2 silicone plates (see Table of Materials). After, under sterile conditions, remove the silicone plates from the autoclave bag and place them in 150 mm plates.
      2. Combine 130 µL of fibronectin (100x, see Table of Materials) with 13 mL of sterile PBS in a 15 mL conical tube. Invert the tube several times to mix.
      3. Add 400 µL of fibronectin solution to each well of the silicone plates. Incubate the plates at 37 °C for a minimum of 2 h or overnight.
      4. Prior to use, aspirate the fibronectin solution and let the plates air-dry in the cell culture hood for at least 30 min to let the fibronectin solution completely evaporate.
         NOTE: The fibronectin solution can be removed from the wells ahead of time, and the now-coated plates can sit at 37 °C for up to a couple of weeks until use.
   2. Prepare the MSC stretch media.
      1. Prepare MSC stretch media by adding 140 µL of ascorbic acid (stock: 100x, final concentration: 50 µg/mL) to 14 mL of pre-warmed MSC media in a 15 mL conical tube.
         NOTE: The ascorbic acid must be added fresh to the MSC media prior to every media change.
2. Seeding of iMSC^SCX+ in the silicone plates
   1. Perform the "Standard lifting cells protocol" (step 1.3).
   2. Resuspend the cells in MSC media and count the viable cells using a hemocytometer or a cell counter.
   3. Calculate the volume of cell suspension needed to obtain 1.25 x 10⁴ cells/cm².
   4. Remove this volume of MSC stretch media from the 15 mL conical tube. Add in the target cells. Invert to homogenous the new cell suspension.
   5. Add 400 µL of the new cell suspension to every well of both silicone plates.
   6. Put the lid back on the 150 mm plates and return them to the incubator at 37 °C for 24 h.
3. Uniaxial tension
   1. The next day, rinse the stretching apparatus (see Table of Materials) with ddH₂O followed by 70% ethanol. Wipe down the apparatus, place it in the cell culture hood, and UV for 15 min.
   2. Retrieve the seeded plates and check the wells for good cell attachment.
   3. Leave one plate in the incubator (static control) and place the second-seeded silicone plate in the stretching apparatus by aligning the screws with the holes in the silicone plate. Replace the lid on the apparatus to ensure sterility.
   4. Attach the apparatus with the seeded silicone plate to the electronic source (see Table of Materials) and place it in the incubator at 37 °C.
   5. On the program, set the cyclic stretching protocol to 4% sinusoidal strain, 0.5 Hz, 2 h per day. Begin the stretch by pressing the start button once. Stretching should begin immediately.
   6. Stretch the cells for a minimum of 3 days. Monitor the cells daily and stop the stretching protocol if cell contraction can be observed. Change the media to fresh MSC stretch media every other day.

Representative Results

Human iPSCs differentiation to iMSCs
As previously described, the current protocol for differentiating iPSCs into iMSCs involves the formation of embryoid bodies². This process takes approximately ten days to induce iMSCs from iPSCs (Figure 1A). However, it is highly recommended to passage the newly generated iMSCs at least twice. This not only helps eliminate the need for gelatin-coated plates but also establishes stable MSC expression. Flow cytometry quantification, conducted after six passages following differentiation, demonstrates a nearly pure cell population with high expression of classic MSC surface markers, including CD44 (83.1%), CD90 (88.4%), and CD105 (99.2%)^17,18 (Figure 1B). In terms of morphology, iMSCs should closely resemble bone marrow MSCs, appearing elongated and fibroblast-like (Figure 1C).

Genetic engineering of iMSCs to overexpress SCX using lentiviral transduction
Lentiviruses were produced by transfecting HEK293T/17 cells with the pLenti-C-mGFP vector, in which SCXB has been inserted to express SCX-GFP⁺, along with two packaging plasmids. Transfections were performed using the BioT method^15,16 with a 1.5:1 ratio of BioT (µl) to DNA (µg).

HEK293T/17 cells were seeded at a density of 5.3 x 10⁴ cells/cm² 18-24 h prior to transfection. At the time of transfection, cells should reach 80%-95% confluency to avoid low-efficiency titers (Figure 2B1, left). Within 24 h of adding the plasmid cocktail, some toxicity was observed, which peaked at 48 h (Figure 2B2). Since the SCX lentiviral vector is GFP-conjugated, GFP expression should be observable after 48 h (Figure 2B3). The presence of high toxicity combined with GFP expression is a reliable indicator of successful transfection. The lentivirus was collected at 48 h and 72 h, and transduction efficiency was assessed using flow cytometry and gene expression analysis. The absolute intensity of GFP-positive cells was used as a proxy for SCX integrations, and it was significantly higher in the higher titers (Figure 3A). The transduction efficiency, measured as the percentage of SCX-GFP⁺ cells, demonstrated a dose-response effect based on the lentiviral load (Figure 3B). Flow cytometry of the iMSC^SCX+ at different passages also showed high stability, with no changes in transgene expression levels or the proportion of transduced cells (Figure 3C). Additionally, after 4 weeks of regular culture without sorting, the iMSC^SCX+ maintained stable overexpression of SCX (Figure 3D). Large-scale transduction was conducted based on the titer, using 75% lentivirus (Figure 2C).

Mechanical loading of iMSC^SCX+
The iMSC^SCX+ cells were seeded onto deformable silicone plates at a cell density of 1.25 x 10⁴ cells/cm² (Figure 4A,B) to allow for cell attachment before initiating the stretching protocol. The final seeding density was optimized to prevent monolayer overgrowth. It's worth noting that excessively high seeding densities resulted in premature cell contraction and early cell death (Figure 4D). For the static control group, cells were plated in identical plates but without undergoing any stretching. The iMSC^SCX+ cells were subjected to stretching in a 2D bioreactor for a minimum of three days, up to seven days, at 4% uniaxial strain and 0.5 Hz for 2 h per day. This stretching regimen is consistent with what has been described as physiologically relevant⁹. After several days of stretching, some degree of cell organization can be observed compared to the static group, which exhibited random cell organization (Figure 4C). Phalloidin staining of the actin filaments further highlights how the cells seem to grow perpendicular to the direction of stretch, in contrast to the random cell organization observed in the static plates (Figure 4E). To characterize the newly generated iTenocytes, cells were collected at three and seven days for gene expression analysis, as previously reported¹⁴. Gene expression analysis reveals that the iMSC^SCX+ cells are mechanoresponsive, as there is a significant upregulation in tenogenic genes (SCX, THBS4, COL1a1, BGN, MKX, and TPPP3)^5,7,19,20 at both three and seven days, compared to just the iMSCs at day 0 (Figure 5A). Additionally, collagen deposition in the media after 7 days of stretching was significantly higher in the iMSC^SCX+ stretched group compared to all other groups (Figure 5B).

Figure 1: iMSC differentiation schematic and flow cytometry characterization. (A) Overall schematic of iTenocyte generation and timeline for iMSC induction. Reproduced with permission from Papalamprou A. et al.¹⁴. (B) Flow cytometry quantification shows a high percent of cells expressing classic MSC surface markers for iPSC-derived MSCs following 6 passages after differentiation. Adapted with permission from Sheyn D. et al.². (C) Phase contrast images of cells after differentiation demonstrate fibroblast-like morphology. Scale bar = 200 µm. Please click here to view a larger version of this figure.

Figure 2: iMSC^SCX+ production schematic. (A) Overall schematic of transfection using 2^nd generation SCX lentivirus vector and transduction of iMSCs. Reproduced with permission from Papalamprou A. et al.¹⁴. (B1) HEK293T/17 cells prior to the addition of plasmid cocktail. (B2) HEK293T/17 cells, after 48 h, express GFP and display toxicity. (B3) Expression of GFP that can be observed in HEK293T/17 cells indicates successful transfection. (C) Generated iMSC^SCX+ cells (with 75% titer) express SCX-GFP⁺ in the nuclei. Scale bars = 400 µm. Please click here to view a larger version of this figure.

Figure 3: Determining the transduction efficiency with flow cytometry and gene expression. The absolute intensity of GFP-positive cells was used as a proxy for SCX integrations using flow cytometry. Vector titers were validated by flow cytometry analysis for all cells to assess lentiviral MOI. (A) Absolute fluorescence per virus titer. (B) Transduction efficiency evaluated as the percentage of SCX-GFP⁺ cells. A dose-response effect of lentiviral load in transduction efficiency was observed when flow results were presented as a percentage of GFP+ cells. MOI, multiplicity of infection, n = 3 independent transductions. One-way ANOVA was used to compare titers; data are mean ± SD; *p < 0.05. (C) Flow cytometry of iMSC^SCX+ after several rounds of passaging indicates no change in the level of stable transgene expression and no change in the proportion of transduced cells. Note that iMSCs transduced with 100% titers stopped dividing at P3. (D) iMSCs transduced with SCX-GFP⁺ lentivirus vector (75%, MOI = 2.9 e5 TU/mL) and assessed the gene expression of SCX at 4 weeks of regular culture without sorting. SCX expression was significantly upregulated in iMSC^SCX+ showing stable overexpression of SCX after 4 weeks. TU, transduction units; data is mean ± SD, **p > 0.01. Reproduced with permission from Papalamprou A. et al.¹⁴. Please click here to view a larger version of this figure.

Figure 4: iMSC^SCX+ seeded into a 2D bioreactor and undergoes cyclic stretching. (A) 2D bioreactor schematic. Reproduced with permission from Papalamprou A. et al.¹². (B) Cells are first seeded into flexible silicone plates and are incubated at 37 °C to allow for attachment prior to cyclic stretching in the 2D bioreactor. (C) iMSC^SCX+ were stretched in a 2D bioreactor for up to 7 days. For static controls, cells were plated in identical plates but with no stretching. (C1) Static control plate after 7 days exhibits stochastic arrangement of cells. (C2) Stretched plate after 7 days shows relatively more cell organization. (D) Three examples of what should not be observed. When the cell seeding density is too high or the cells have been stretched for too many days, cells begin to detach. (D1) Cells are only slightly overgrown. Yellow arrows indicate the beginning of premature cell contraction. (D2) Moderate level of overgrowth. Cells begin to detach from the plate. (D3) Severe level of overgrowth. Cells are no longer growing in monolayer and have formed 3D structures. Black scale bars = 400 µm. White scale bars = 1000 µm. (E) After 7 days of stretching (or in culture for the static plate), cells were fixed with phalloidin for actin filaments (red) and counterstained with DAPI for nuclei (blue). White scale bars = 200 µm. Please click here to view a larger version of this figure.

Figure 5: Stimulation of tenogenic marker gene expression by Scx overexpression and uniaxial stretch in a 2D bioreactor. (A) iMSC^SCX+ were stretched in a 2D bioreactor for 7 days. For static controls, cells were plated in identical plates but with no stretching. Gene expression analyses reveal that iMSC^SCX+ is mechanoresponsive. ND = no detection. One-way ANOVA was used to compare gene expression at each timepoint vs. d0. N = 8/group. (B) Collagen deposition following 7 days of stretch in the 2D bioreactor. Data are mean ± SD; n = 8/group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Reproduced with permission from Papalamprou A. et al.¹². Please click here to view a larger version of this figure.

Discussion

In this protocol, iTenocytes are generated through three main steps: (1) induction of iPSCs to iMSCs, (2) overexpression of SCX using a lentiviral vector, and (3) maturation of cells through 2D uniaxial tension.

The protocol presented for differentiating iPSCs into iMSCs has been previously described by our group². Since that publication, numerous protocols have been developed, including an established protocol for using iMSCs in clinical trials^21,22,23, as well as commercially available differentiation kits. A review of the trilineage potential of iMSCs has also been investigated previously². While methodologies differ, all protocols emphasize the need for post-differentiation expansion of iMSCs for several passages to ensure stable expression of MSC surface markers. At this stage, using a 1:3 split ratio at approximately 70% confluence will result in a relatively confluent plate within 4-6 days after passaging.

When selecting the optimal titer for transduction, it's crucial to strike a balance between cell viability and efficiency. While the iMSCs transduced with the 100% titer may show the highest level of SCX-GFP positive cells, it's worth noting that these cells ceased dividing three passages after transduction (Figure 3C), possibly due to DNA-induced toxicity. Therefore, it is advisable to use a titer less than 100%. Before seeding the silicone plates, it's recommended to reassess the SCX-GFP levels using flow cytometry. If the data indicates that the efficiency is below the desired threshold, sorting the iMSC^SCX+ cells before seeding is advisable, particularly for in vivo applications.

Once the iMSC^SCX+ cells have been seeded in the silicone plates, they must be incubated at 37 °C overnight to allow for cell attachment before stretching. The described cell density of 1.25 x 10⁴ cells/cm² in the silicone plates was optimized to prevent monolayer overgrowth, which can contribute to static tension²⁴. Additionally, studies suggest that direct cell-to-cell contact in confluent cultures in substrates of varying stiffness can alter cell behavior^24,25,26. During pilot experiments, some visible cell detachment from the silicone plates was observed, particularly at later timepoints (Figure 4D). This could be attributed to the formation of ECM cell sheets due to overconfluence²⁴. Therefore, critical parameters include cell density and the number of stretch bouts. In the current methods, cells were collected at days 3 and 7 for the assessment of tenogenic potential via gene expression analysis. However, it is recommended that cells be stretched in the 2D bioreactor for at least three days, with subsequent monitoring of stretched and static plates to prevent overgrowth and cell detachment.

After several stretch bouts, a degree of cell alignment and organization can be observed (Figure 4C1,E). This aligns with many studies where cell alignment perpendicular to the axis of strain is observed in response to uniaxial strain in vitro^24,27. In comparison, the static plate displays stochastic cell organization (Figure 4C2,E).

To the best of our knowledge, only a few studies have explored the synergistic effects of SCX overexpression and mechanical stimulation for the differentiation of MSCs into tenocytes or ligamentocytes^24,28,29. Gaspar et al. employed a similar 2D bioreactor system as the one described here but applied higher levels of total strain (10% at 1 Hz for 12 h/day). Interestingly, they were unable to detect changes in the expression of SCX, TNMD, and COL1a1 in BM-MSCs and human tenocytes. However, this may be attributed to the higher applied strains used in their study²⁴. Chen et al. used a lentiviral vector to overexpress SCX in hESC-MSCs assembled in multi-layered sheets. They applied uniaxial cyclic load (10% strain at 1 Hz for 2 h/day for up to 21 days) and observed upregulation of COL1a1, COL1a2, COL14, and TNMD, as well as increased ECM deposition²⁹. Nichols et al. transiently transfected C3H10T1/2 cells with full-length murine Scx cDNA and cultured the cells in 3D collagen hydrogels under uniaxial cyclic strain (1%, 1 Hz, 30 min/day for up to 14 days). Similar to our findings, their group observed elevated expression of SCX and COL1A1 in the strained and overexpressed constructs but found no change in TNMD expression in response to cyclic stretching²⁸.

Additionally, it might be intriguing to consider the overexpression of other tendon-related markers like MKX. Tsutsumi et al. explored the combined effect of overexpressing MKX in C3H10T1/2 cells and subjecting them to cyclic mechanical stretching in a 3D system. They demonstrated a significant upregulation of SCX, COL1a1, DCN, and COL3a1, along with the alignment of collagen fibril bundles and actin filaments³⁰.

It's important to acknowledge that this method for generating iTenocytes has its limitations. While the commercially available 2D bioreactor is advantageous for proof-of-concept work, its size restricts the yield. If these cells are needed for high-throughput assays or as a potential off-the-shelf cell source for tendon repair therapies, exploring systems capable of implementing uniaxial stretching on a larger scale should be considered. Moreover, further investigations should encompass the expansion of iTenocytes to confirm stable tenogenic expression, and assessing their contribution to in vivo regeneration is crucial for evaluating their tenogenic potential.

Materials

Name	Company	Catalog Number	Comments
2-mercaptoethanol	Sigma Aldrich	M3148
Accutase	StemCell Technologies	7920	cell dissociation reagent
Antibiotic-antimycotic solution	Thermofisher	15240096
Anti-CD105	Ancell	326-050
APC mouse anti-human CD44	BD Biosciences	559942
APC mouse IgG2 K isotype control	BD Biosciences	555745
BenchMark fetal bovine serum	GeminiBio	100-106
Biglycan	Thermofisher	Hs00959143_m1
Bovine serum albumin	Millipore Sigma	A3733
Collagen type I alpha 1 chain human Taqman primer	Thermofisher	Hs00164004_m1
Collagen type III alpha 1 chain human Taqman primer	Thermofisher	Hs00943809_m1
Dimethyl sulfoxide	Millipore Sigma	D8418
DMEM, low glucose, pyruvate, no glutamine, no phenol red	Thermofisher	11054020
Eagle's minimum essential medium (EMEM)	ATCC	30-2003
Fibronectin bovine plasma	Sigma Aldrich	F1141
FITC mouse anti-human CD90	BD Biosciences	555595
Gelatin from porcine skin	Sigma Aldrich	G1890
Goat anti Mouse IgG1-PE	Bio-Rad	STAR117
HEK 293T/17	ATCC	CRL-11268
IMDM, no phenol red	Thermofisher	21056023
iPSCs: 83i-cntr-33n1	Cedars-Sinai iPSC Core Facility	N/A	https://biomanufacturing.cedars-sinai.org/product/cs83ictr-33nxx/
Isotype Control Antibody, mouse IgG2a-FITC	Miltenyi Biotec	130-113-271
KnockOut serum replacement	Thermofisher	10828010
L-ascorbic acid	Sigma Aldrich	A4544
L-Glutamine	Thermofisher	2503081
Matrigel	Corning	354230	basement membrane matrix
MechanoCulture FX	CellScale	N/A	stretching apparatus
MEM non-essential amino acids solution	Thermofisher	11140050
Mohawk human Taqman primer	Thermofisher	Hs00543190_m1
mTeSR Plus	StemCell Technologies	100-0276
PBS	Thermofisher	10010023
Platelet-derived growth factor receptor A human Taqman primer	Thermofisher	Hs00998018_m1
Poly(2-hydroxyethyl methacrylate)	Sigma Aldrich	192066
Polybrene infection/transfection reagents	Millipore Sigma	TR-1003
Recombinant human  TGF-beta 1 protein human Taqman primer	RnD Systems	240-B
Scleraxis human Taqman primer	Thermofisher	Hs03054634_g1
SCXA (SCX) (NM_00108050514) human tagged ORF clone	OriGene	RC224305L4
Silicone plates	CellScale	N/A
Sodium azide	Millipore Sigma	S2002
Tenascin C human Taqman primer	Thermofisher	Hs00370384_m1
Tenomodulin human Taqman primer	Thermofisher	Hs00223332_m1
Thrombospondin 4 human Taqman primer	Thermofisher	Hs00170261_m1
Transfection reagent, BioT	Bioland Scientific LLC	B01-01
Trypsin-EDTA (0.25%)	Thermofisher	25200072
Tubulin polymerization promoting protein family member 3	Thermofisher	Hs03043892_m1
Y-27632 dihydrochloride	Biogems	1293823