Name: Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension (Video) | JoVE Journal
Uploaded: 2024-03-01T13:10:00.000+00:00
Duration: 4 min 48 s
Description: Learn to generate iTenocytes from iPSC-derived mesenchymal stromal cells using Scleraxis overexpression and 2D uniaxial stretching. This method enhances tendon-related gene expression for improved tendon repair strategies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This study was partially supported by the NIH/NIAMS K01AR071512 and CIRM DISC0-14350 to Dmitriy Sheyn. The two lentivirus packaging plasmids were a gift from the Simon Knott laboratory (Department of Biomedical Sciences, Cedars-Sinai Medical Center).

Harnessing iPSC-Derived iTenocytes for Effective Cell-Based Therapy

<ol><li>Lim, W. L., Liau, L. L., Ng, M. H., Chowdhury, S. R., Law, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+progress+in+tendon+and+ligament+tissue+engineering%5BTitle%5D)+AND+%22Tissue+Engineering+and+Regenerative%22%5BJournal%5D)">Current progress in tendon and ligament tissue engineering.</a> Tissue Engineering and Regenerative. 16 (6), 549-571 (2019).</li>
<li>Sheyn, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+induced+pluripotent+stem+cells+differentiate+into+functional+mesenchymal+stem+cells+and+repair+bone+defects%5BTitle%5D)+AND+%22Stem+Cells+Translational+Medicine%22%5BJournal%5D)">Human induced pluripotent stem cells differentiate into functional mesenchymal stem cells and repair bone defects.</a> Stem Cells Translational Medicine. 5 (11), 1447-1460 (2016).</li>
<li>Czaplewski, S. K., Tsai, T. L., Duenwald-Kuehl, S. E., Vanderby, R., Li, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tenogenic+differentiation+of+human+induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells+dictated+by+properties+of+braided+submicron+fibrous+scaffolds%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Tenogenic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells dictated by properties of braided submicron fibrous scaffolds.</a> Biomaterials. 35 (25), 6907-6917 (2014).</li>
<li>Jo, C. H., Lim, H. J., Yoon, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+tendon-specific+markers+in+various+human+tissues%2C+tenocytes+and+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Tissue+Engineering+and+Regenerative%22%5BJournal%5D)">Characterization of tendon-specific markers in various human tissues, tenocytes and mesenchymal stem cells.</a> Tissue Engineering and Regenerative. 16 (2), 151-159 (2019).</li>
<li>Shukunami, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scleraxis+is+a+transcriptional+activator+that+regulates+the+expression+of+Tenomodulin%2C+a+marker+of+mature+tenocytes+and+ligamentocytes%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Scleraxis is a transcriptional activator that regulates the expression of Tenomodulin, a marker of mature tenocytes and ligamentocytes.</a> Scientific Reports. 8 (1), 3155(2018).</li>
<li>Huang, A. H., Lu, H. H., Schweitzer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+regulation+of+tendon+cell+fate+during+development%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">Molecular regulation of tendon cell fate during development.</a> Journal of Orthopaedic Research. 33 (6), 800-812 (2015).</li>
<li>Harvey, T., Flamenco, S., Fan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Tppp3%28%2B%29Pdgfra%28%2B%29+tendon+stem+cell+population+contributes+to+regeneration+and+reveals+a+shared+role+for+PDGF+signalling+in+regeneration+and+fibrosis%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">A Tppp3(+)Pdgfra(+) tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis.</a> Nature Cell Biology. 21 (12), 1490-1503 (2019).</li>
<li>Yang, G., Rothrauff, B. B., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tendon+and+ligament+regeneration+and+repair%3A+clinical+relevance+and+developmental+paradigm%5BTitle%5D)+AND+%22Birth+Defects+Research+Part+C%3A+Embryo+Today%22%5BJournal%5D)">Tendon and ligament regeneration and repair: clinical relevance and developmental paradigm.</a> Birth Defects Research Part C: Embryo Today. 99 (3), 203-222 (2013).</li>
<li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+loading+models+for+tendon+mechanobiology%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">In vitro loading models for tendon mechanobiology.</a> Journal of Orthopaedic Research. 36 (2), 566-575 (2018).</li>
<li>Wu, S. Y., Kim, W., Kremen, T. J. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+cellular+strain+models+of+tendon+biology+and+tenogenic+differentiation%5BTitle%5D)+AND+%22Frontiers+in+Bioengineering+and+Biotechnology%22%5BJournal%5D)">In vitro cellular strain models of tendon biology and tenogenic differentiation.</a> Frontiers in Bioengineering and Biotechnology. 10, 826748(2022).</li>
<li>Screen, H. R., Lee, D. A., Bader, D. L., Shelton, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+investigation+into+the+effects+of+the+hierarchical+structure+of+tendon+fascicles+on+micromechanical+properties%5BTitle%5D)+AND+%22Proceedings+of+the+Institution+of+Mechanical+Engineers%2C+Part+H%22%5BJournal%5D)">An investigation into the effects of the hierarchical structure of tendon fascicles on micromechanical properties.</a> Proceedings of the Institution of Mechanical Engineers, Part H. 218 (2), 109-119 (2004).</li>
<li>Tatullo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+influence+of+tissue+culture+plates+and+extracellular+matrix+on+mesenchymal+stem+cell+behavior%3A+A+topical+review%5BTitle%5D)+AND+%22International+Journal+of+Immunopathology+and+Pharmacology%22%5BJournal%5D)">Mechanical influence of tissue culture plates and extracellular matrix on mesenchymal stem cell behavior: A topical review.</a> International Journal of Immunopathology and Pharmacology. 29 (1), 3-8 (2016).</li>
<li>Ding, S., Kingshott, P., Thissen, H., Pera, M., Wang, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+human+mesenchymal+and+pluripotent+stem+cell+behavior+using+biophysical+and+biochemical+cues%3A+A+review%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">Modulation of human mesenchymal and pluripotent stem cell behavior using biophysical and biochemical cues: A review.</a> Biotechnology and Bioengineering. 114 (2), 260-280 (2017).</li>
<li>Papalamprou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directing+iPSC+differentiation+into+iTenocytes+using+combined+scleraxis+overexpression+and+cyclic+loading%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">Directing iPSC differentiation into iTenocytes using combined scleraxis overexpression and cyclic loading.</a> Journal of Orthopaedic Research. 41 (6), 1148-1161 (2023).</li>
<li>Pryce, B. A., Brent, A. E., Murchison, N. D., Tabin, C. J., Schweitzer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+transgenic+tendon+reporters%2C+ScxGFP+and+ScxAP%2C+using+regulatory+elements+of+the+scleraxis+gene%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">Generation of transgenic tendon reporters, ScxGFP and ScxAP, using regulatory elements of the scleraxis gene.</a> Developmental Dynamics. 236 (6), 1677-1682 (2007).</li>
<li>Stewart, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lentivirus-delivered+stable+gene+silencing+by+RNAi+in+primary+cells%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">Lentivirus-delivered stable gene silencing by RNAi in primary cells.</a> RNA. 9 (4), 493-501 (2003).</li>
<li>Mendicino, M., Bailey, A. M., Wonnacott, K., Puri, R. K., Bauer, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MSC-based+product+characterization+for+clinical+trials%3A+an+FDA+perspective%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">MSC-based product characterization for clinical trials: an FDA perspective.</a> Cell Stem Cell. 14 (2), 141-145 (2014).</li>
<li>Bourin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stromal+cells+from+the+adipose+tissue-derived+stromal+vascular+fraction+and+culture+expanded+adipose+tissue-derived+stromal%2Fstem+cells%3A+a+joint+statement+of+the+International+Federation+for+Adipose+Therapeutics+and+Science+%28IFATS%29+and+the+International+Society+for+Cellular+Therapy+%28ISCT%29%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT).</a> Cytotherapy. 15 (6), 641-648 (2013).</li>
<li>Dunkman, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+tendon+injury+response+is+influenced+by+decorin+and+biglycan%5BTitle%5D)+AND+%22Annals+of+Biomedical+Engineering%22%5BJournal%5D)">The tendon injury response is influenced by decorin and biglycan.</a> Annals of Biomedical Engineering. 42 (3), 619-630 (2014).</li>
<li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crucial+transcription+factors+in+tendon+development+and+differentiation%3A+their+potential+for+tendon+regeneration%5BTitle%5D)+AND+%22Cell+and+Tissue+Research%22%5BJournal%5D)">Crucial transcription factors in tendon development and differentiation: their potential for tendon regeneration.</a> Cell and Tissue Research. 356 (2), 287-298 (2014).</li>
<li>Bloor, A. J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production%2C+safety+and+efficacy+of+iPSC-derived+mesenchymal+stromal+cells+in+acute+steroid-resistant+graft+versus+host+disease%3A+a+phase+I%2C+multicenter%2C+open-label%2C+dose-escalation+study%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Production, safety and efficacy of iPSC-derived mesenchymal stromal cells in acute steroid-resistant graft versus host disease: a phase I, multicenter, open-label, dose-escalation study.</a> Nature Medicine. 26 (11), 1720-1725 (2020).</li>
<li>McGrath, M., Tam, E., Sladkova, M., AlManaie, A., Zimmer, M., de Peppo, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GMP-compatible+and+xeno-free+cultivation+of+mesenchymal+progenitors+derived+from+human-induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Stem+Cell+Research+and+Therapy%22%5BJournal%5D)">GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells.</a> Stem Cell Research and Therapy. 10 (1), 11(2019).</li>
<li>Lian, Q., Zhang, Y., Liang, X., Gao, F., Tse, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+differentiation+of+human-induced+pluripotent+stem+cells+to+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Directed differentiation of human-induced pluripotent stem cells to mesenchymal stem cells.</a> Methods in Molecular Biology. 1416, 289-298 (2016).</li>
<li>Gaspar, D., Ryan, C. N. M., Zeugolis, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multifactorial+bottom-up+bioengineering+approaches+for+the+development+of+living+tissue+substitutes%5BTitle%5D)+AND+%22FASEB+Journal%22%5BJournal%5D)">Multifactorial bottom-up bioengineering approaches for the development of living tissue substitutes.</a> FASEB Journal. 33 (4), 5741-5754 (2019).</li>
<li>Mao, A. S., Shin, J. W., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+substrate+stiffness+and+cell-cell+contact+on+mesenchymal+stem+cell+differentiation%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Effects of substrate stiffness and cell-cell contact on mesenchymal stem cell differentiation.</a> Biomaterials. 98, 184-191 (2016).</li>
<li>Tilghman, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PLoS One. 5 (9), e12905(2010).</li>
<li>Chagnon-Lessard, S., Jean-Ruel, H., Godin, M., Pelling, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+orientation+is+guided+by+strain+gradients%5BTitle%5D)+AND+%22Integrative+Biology+%28United+Kingdom%29%22%5BJournal%5D)">Cellular orientation is guided by strain gradients.</a> Integrative Biology (United Kingdom). 9 (7), 607-618 (2017).</li>
<li>Nichols, A. E. C., Werre, S. R., Dahlgren, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+scleraxis+overexpression+combined+with+cyclic+strain+enhances+ligament+cell+differentiation%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">Transient scleraxis overexpression combined with cyclic strain enhances ligament cell differentiation.</a> Tissue Engineering Part A. 24 (19-20), 1444-1455 (2018).</li>
<li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Force+and+scleraxis+synergistically+promote+the+commitment+of+human+ES+cells+derived+MSCs+to+tenocytes%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Force and scleraxis synergistically promote the commitment of human ES cells derived MSCs to tenocytes.</a> Scientific Reports. 2, 977(2012).</li>
<li>Kataoka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+neo-genesis+of+tendon%2Fligament-like+tissue+by+combination+of+mohawk+and+a+three-dimensional+cyclic+mechanical+stretch+culture+system%5BTitle%5D)+AND+%22Frontiers+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">In vitro neo-genesis of tendon/ligament-like tissue by combination of mohawk and a three-dimensional cyclic mechanical stretch culture system.</a> Frontiers in Cell and Developmental Biology. 8, 307(2020).</li>
</ol>

All authors have no conflicts of interest to disclose.

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 trials21,22,23, 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&#39;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&#39;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&#39;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 iMSCSCX+ cells before seeding is advisable, particularly for in vivo applications.
Once the iMSCSCX+ cells have been seeded in the silicone plates, they must be incubated at 37 &#176;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 behavior24,25,26. 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 vitro24,27. 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 ligamentocytes24,28,29. 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&#39;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.

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.

<table><tbody><tr><td>2-mercaptoethanol&amp;nbsp;</td><td>Sigma Aldrich</td><td>M3148</td><td></td></tr><tr><td>Accutase</td><td>StemCell Technologies</td><td>7920</td><td>cell dissociation reagent</td></tr><tr><td>Antibiotic-antimycotic solution</td><td>Thermofisher</td><td>15240096</td><td></td></tr><tr><td>Anti-CD105</td><td>Ancell</td><td>326-050</td><td></td></tr><tr><td>APC mouse anti-human CD44</td><td>BD Biosciences</td><td>559942</td><td></td></tr><tr><td>APC mouse IgG2 K isotype control</td><td>BD Biosciences</td><td>555745</td><td></td></tr><tr><td>BenchMark fetal bovine serum</td><td>GeminiBio</td><td>100-106</td><td></td></tr><tr><td>Biglycan</td><td>Thermofisher</td><td>Hs00959143_m1</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Millipore Sigma</td><td>A3733</td><td></td></tr><tr><td>Collagen type I alpha 1 chain human Taqman primer</td><td>Thermofisher</td><td>Hs00164004_m1</td><td></td></tr><tr><td>Collagen type III alpha 1 chain human Taqman primer</td><td>Thermofisher</td><td>Hs00943809_m1</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Millipore Sigma</td><td>D8418</td><td></td></tr><tr><td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td><td>Thermofisher</td><td>11054020</td><td></td></tr><tr><td>Eagle&#039;s minimum essential medium (EMEM)</td><td>ATCC</td><td>30-2003</td><td></td></tr><tr><td>Fibronectin bovine plasma</td><td>Sigma Aldrich</td><td>F1141</td><td></td></tr><tr><td>FITC mouse anti-human CD90</td><td>BD Biosciences</td><td>555595</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma Aldrich</td><td>G1890</td><td></td></tr><tr><td>Goat anti Mouse IgG1-PE</td><td>Bio-Rad</td><td>STAR117</td><td></td></tr><tr><td>HEK 293T/17</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>IMDM, no phenol red</td><td>Thermofisher</td><td>21056023</td><td></td></tr><tr><td>iPSCs: 83i-cntr-33n1</td><td>Cedars-Sinai iPSC Core Facility</td><td>N/A</td><td>https://biomanufacturing.cedars-sinai.org/product/cs83ictr-33nxx/</td></tr><tr><td>Isotype Control Antibody, mouse IgG2a-FITC</td><td>Miltenyi Biotec</td><td>130-113-271</td><td></td></tr><tr><td>KnockOut serum replacement</td><td>Thermofisher</td><td>10828010</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma Aldrich</td><td>A4544</td><td></td></tr><tr><td>L-Glutamine</td><td>Thermofisher</td><td>2503081</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354230</td><td>basement membrane matrix</td></tr><tr><td>MechanoCulture FX</td><td>CellScale</td><td>N/A</td><td>stretching apparatus</td></tr><tr><td>MEM non-essential amino acids solution</td><td>Thermofisher</td><td>11140050</td><td></td></tr><tr><td>Mohawk human Taqman primer</td><td>Thermofisher</td><td>Hs00543190_m1</td><td></td></tr><tr><td>mTeSR Plus</td><td>StemCell Technologies</td><td>100-0276</td><td></td></tr><tr><td>PBS</td><td>Thermofisher</td><td>10010023</td><td></td></tr><tr><td>Platelet-derived growth factor receptor A human Taqman primer</td><td>Thermofisher</td><td>Hs00998018_m1</td><td></td></tr><tr><td>Poly(2-hydroxyethyl methacrylate)</td><td>Sigma Aldrich</td><td>192066</td><td></td></tr><tr><td>Polybrene infection/transfection reagents</td><td>Millipore Sigma</td><td>TR-1003</td><td></td></tr><tr><td>Recombinant human&amp;nbsp; TGF-beta 1 protein human Taqman primer</td><td>RnD Systems</td><td>240-B</td><td></td></tr><tr><td>Scleraxis human Taqman primer</td><td>Thermofisher</td><td>Hs03054634_g1</td><td></td></tr><tr><td>SCXA (SCX) (NM_00108050514) human tagged ORF clone</td><td>OriGene</td><td>RC224305L4</td><td></td></tr><tr><td>Silicone plates</td><td>CellScale</td><td>N/A</td><td></td></tr><tr><td>Sodium azide</td><td>Millipore Sigma</td><td>S2002</td><td></td></tr><tr><td>Tenascin C human Taqman primer</td><td>Thermofisher</td><td>Hs00370384_m1</td><td></td></tr><tr><td>Tenomodulin human Taqman primer</td><td>Thermofisher</td><td>Hs00223332_m1</td><td></td></tr><tr><td>Thrombospondin 4 human Taqman primer</td><td>Thermofisher</td><td>Hs00170261_m1</td><td></td></tr><tr><td>Transfection reagent, BioT</td><td>Bioland Scientific LLC</td><td>B01-01</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Thermofisher</td><td>25200072</td><td></td></tr><tr><td>Tubulin polymerization promoting protein family member 3</td><td>Thermofisher</td><td>Hs03043892_m1</td><td></td></tr><tr><td>Y-27632 dihydrochloride</td><td>Biogems</td><td>1293823</td><td></td></tr></tbody></table>

generation of induced pluripotent stem cell-derived itenocytes via combined scleraxis overexpression and 2d uniaxial tension

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

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.

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 method15,16 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 iMSCSCX+ 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 iMSCSCX+ maintained stable overexpression of SCX (Figure 3D). Large-scale transduction was conducted based on the titer, using 75% lentivirus (Figure 2C).
Mechanical loading of iMSCSCX+ 
The iMSCSCX+ 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 iMSCSCX+ 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 iMSCSCX+ 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 iMSCSCX+ stretched group compared to all other groups (Figure 5B).
<img alt="iPSC differentiation process, including CD44/CD105/CD90 analysis; 2D bioreactor and cell expansion." class="xfigimg" src="/files/ftp_upload/65837/65837fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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. <a href="https://www.jove.com/files/ftp_upload/65837/65837fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lentiviral transfection process diagram with iPSC differentiation into iTenocytes; GFP fluorescence image." class="xfigimg" src="/files/ftp_upload/65837/65837fig02v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: iMSCSCX+ production schematic. (A) Overall schematic of transfection using 2nd 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 iMSCSCX+ cells (with 75% titer) express SCX-GFP+ in the nuclei. Scale bars = 400 µm. <a href="https://www.jove.com/files/ftp_upload/65837/65837fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Gene editing efficacy analysis; bar, violin plots, flow cytometry; vector titration; GFP fluorescence." class="xfigimg" src="/files/ftp_upload/65837/65837fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
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 &lt; 0.05. (C) Flow cytometry of iMSCSCX+ 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 iMSCSCX+ showing stable overexpression of SCX after 4 weeks. TU, transduction units; data is mean ± SD, **p &gt; 0.01. Reproduced with permission from Papalamprou A. et al.14. <a href="https://www.jove.com/files/ftp_upload/65837/65837fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="iMSC to iTenocytes via 2D bioreactor. Microscopy shows static/stretched culture effects." class="xfigimg" src="/files/ftp_upload/65837/65837fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: iMSCSCX+ 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) iMSCSCX+ 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. <a href="https://www.jove.com/files/ftp_upload/65837/65837fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar graph analysis of gene expression in iMSC cells; SCX, THBS4, BGN, protein levels, experimental chart." class="xfigimg" src="/files/ftp_upload/65837/65837fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Stimulation of tenogenic marker gene expression by Scx overexpression and uniaxial stretch in a 2D bioreactor. (A) iMSCSCX+ 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 iMSCSCX+ 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 &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Reproduced with permission from Papalamprou A. et al.12. <a href="https://www.jove.com/files/ftp_upload/65837/65837fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

generation-induced-pluripotent-stem-cell-derived-itenocytes-via

Research

JoVE Journal

Bioengineering

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells.  Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28.  Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation of Mice Derived from Induced Pluripotent Stem Cells

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Learn to generate iTenocytes from iPSC-derived mesenchymal stromal cells using Scleraxis overexpression and 2D uniaxial stretching. This method enhances tendon-related gene expression for improved tendon repair strategies.

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

Explore More Videos