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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+progress+in+tendon+and+ligament+tissue+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+differentiate+into+functional+mesenchymal+stem+cells+and+repair+bone+defects.">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. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+tendon-specific+markers+in+various+human+tissues,+tenocytes+and+mesenchymal+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scleraxis+is+a+transcriptional+activator+that+regulates+the+expression+of+Tenomodulin,+a+marker+of+mature+tenocytes+and+ligamentocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+tendon+cell+fate+during+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tppp3(+)Pdgfra(+)+tendon+stem+cell+population+contributes+to+regeneration+and+reveals+a+shared+role+for+PDGF+signalling+in+regeneration+and+fibrosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+and+ligament+regeneration+and+repair:+clinical+relevance+and+developmental+paradigm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+loading+models+for+tendon+mechanobiology.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cellular+strain+models+of+tendon+biology+and+tenogenic+differentiation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+into+the+effects+of+the+hierarchical+structure+of+tendon+fascicles+on+micromechanical+properties.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+influence+of+tissue+culture+plates+and+extracellular+matrix+on+mesenchymal+stem+cell+behavior:+A+topical+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+human+mesenchymal+and+pluripotent+stem+cell+behavior+using+biophysical+and+biochemical+cues:+A+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+iPSC+differentiation+into+iTenocytes+using+combined+scleraxis+overexpression+and+cyclic+loading.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transgenic+tendon+reporters,+ScxGFP+and+ScxAP,+using+regulatory+elements+of+the+scleraxis+gene.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-delivered+stable+gene+silencing+by+RNAi+in+primary+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC-based+product+characterization+for+clinical+trials:+an+FDA+perspective.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tendon+injury+response+is+influenced+by+decorin+and+biglycan.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+transcription+factors+in+tendon+development+and+differentiation:+their+potential+for+tendon+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMP-compatible+and+xeno-free+cultivation+of+mesenchymal+progenitors+derived+from+human-induced+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human-induced+pluripotent+stem+cells+to+mesenchymal+stem+cells.">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. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+orientation+is+guided+by+strain+gradients.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+scleraxis+overexpression+combined+with+cyclic+strain+enhances+ligament+cell+differentiation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+scleraxis+synergistically+promote+the+commitment+of+human+ES+cells+derived+MSCs+to+tenocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+neo-genesis+of+tendon/ligament-like+tissue+by+combination+of+mohawk+and+a+three-dimensional+cyclic+mechanical+stretch+culture+system.">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</sup...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol&#160;</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&#39;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&#160; 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

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.

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.

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 cy...

Watch this Scientific Journal Video about Advancements in Cell and Tissue Engineering for Tendon Repair at JoVE.com

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 ...

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JoVE Journal

Bioengineering

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

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

1.2K Views.  Cedars-Sinai Medical Center.  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.

Harnessing iPSC-Derived iTenocytes for Effective Cell-Based Therapy

Summary