Directing iPSC Differentiation into iTenocytes using Combined Scleraxis Overexpression and Cyclic Loading

Regenerative therapies for tendon are falling behind other tissues due to the lack of an appropriate and potent cell therapeutic candidate. This study aimed to induce cell tenogenesis using stable Scleraxis (Scx) overexpression in combination with uniaxial mechanical stretch of mesenchymal stromal cells (MSCs) of different origins. Scleraxis (Scx) is the single direct molecular regulator of tendon differentiation known to date. Mechanoregulation is known to be a central element guiding tendon development and healing. Cells explored were bone marrow-derived (BM-)MSCs as well as MSCs differentiated from induced pluripotent stem cells (iMSCs). Mechanical stimulation combined with Scx overexpression resulted in morphometric and cytoskeleton-related changes, upregulation of early and late tendon markers, increased ECM deposition and alignment, and tenomodulin perinuclear localization in iMSCs, which was greater compared to BM-MSCs and controls. Our findings suggest that these cells can be differentiated into tenocytes and may be a better candidate for tendon cell therapy applications than BM-MSCs.


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Optical density at 535 nm was determined for samples and assay standards. Collagen 2 3 3 concentration was calculated from the standard curve using regression analysis and plotted in 2 3 4 µg/ml units. All experimental samples (n=8) and assay standards were conducted in technical 2 3 5 triplicates. Cell cultures of iMSC SCX+ were grown in static culture or uniaxially stretched in the 2D 2 3 9 bioreactor for 7 days as described above. After 7 days, cells were briefly washed with PBS and 2 4 0 fixed with 10% buffered formalin for 15 min at RT. Cells were washed again, and nonspecific 2 4 1 sites were blocked with 10% normal donkey serum (Jackson Immunoresearch, West Grove, PA) 2 4 2 in 0.3% v/v PBS-Tween® 20 Detergent (PBS-T) for 45 min. After, cells were incubated at 4°C 2 4 3 overnight with the primary antibody for Tnmd (HPA055634, Sigma, Darmstadt, Germany) or 2 4 4 Collagen-1 (1310001, Bio-Rad). Cells were then washed 3 times with PBS-T, followed by a 2h 2 4 5 incubation at RT with the secondary antibody donkey anti-rabbit Alexa Fluor ® 647-conjugated 2 4 6 Affinipure (Jackson Immunoresearch). Nuclei were counterstained with DAPI. Fluorescent 2 4 7 images were captured with a Revolve fluorescent microscope (Revolve). Images of single 2 4 8 channels were taken, and they were merged either during imaging or using Adobe Photoshop 2 4 9 CS6 post imaging. Adobe Illustrator CS6 was used for final figure production. BioRender 2 5 0 software program was used to prepare the graphical abstract and additional graphics used 2 5 1 throughout the manuscript. All data are presented as mean ± standard deviation from the mean. Normally distributed data 2 5 5 were analyzed with unpaired t-test (for 2 groups), or non-repeated measures analysis of variance 2 5 6 followed by Tukey-Kramer HSD post hoc analysis when more than 2 groups were compared.

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Non-parametric data were analyzed using the Mann-Whitney and Kruskal-Wallis tests. To 2 5 8 compare gene expression levels over time, a mixed repeated measures model was used, followed 2 5 9 by Tukey's post hoc tests for between group comparisons. Lastly, to analyze the orientation of 2 6 0 nuclei and actin filaments between the static and stretched conditions, medians were compared 2 6 1 using the Mann-Whitney test, while the two frequency distributions were compared using the 2 6 2 Kolmogorov-Smirnov test. Statistical significance was set at p<0.05. Statistical analyses were 2 6 3 performed with GraphPad Prism 9.0.

Confirmation of stable lentiviral-driven Scx overexpression in BM-MSC SCX+ and iMSC SCX+ 2 6 7
We generated lentiviral vectors expressing Scx tagged with GFP at the C-terminus and under the 2 6 8 constitutively active CMV promoter, as previously described. 33; 34 The absolute intensity of GFP-2 6 9 positive cells was used as a proxy for Scx integrations using flow cytometry. Absolute GFP 2 7 0 fluorescence was similar for the highest titers (Suppl. Fig. 1A). A dose-response effect of 2 7 1 lentiviral load in transduction efficiency was observed when flow results were presented as 2 7 2 percentage of GFP+ cells (Supp Fig. 1B). We expanded BM-MSCs and iMSCs transduced with 2 7 3 Scx-GFP lentivirus vector (BM-MSC SCX+ and iMSC SCX+ ) and assessed the expression of Scx at 4 2 7 4 weeks of regular culture without sorting. Scx expression was significantly upregulated in BM-2 7 5 MSC SCX+ and iMSC SCX+ showing stable overexpression of Scx after 4 weeks (Suppl. Fig. 1C). 2 7 6 2 7 7

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Cellular morphology and orientation of iMSCs and iMSC SCX+ was examined after 7 days of 3 5 0 uniaxial loading or static culture via phalloidin staining of the actin fibers of the cytoskeleton.

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Actin filaments aligned perpendicular to the axis of the load in the stretched plates, whereas a 3 5 2 stochastic cytoskeleton alignment was observed in the static culture (Fig. 4A). Additionally, an 3 5 3 unbiased morphometric analysis was conducted on both culture conditions in iMSCs and 3 5 4 iMSC SCX+ by using high content image analysis of the nuclei, whereby the operator of the 3 5 5 software was blinded to the groups. Nucleic orientation assessment showed that uniaxial cyclic 3 5 6 loading for 7 days significantly affected the frequency distribution of nuclear orientation in 3 5 7 iMSCs and iMSC SCX+ , which was assessed as an angle from the axis of loading (Fig. 4B). The 3 5 8 medians of nuclear orientation of the static and the stretched conditions were shown to be 3 5 9 significantly different. In iMSCs after 7 days of cyclic stretching, 54% of stretched nuclei were 3 6 0 found at 65-90 o angle compared to 33.7% nuclei in the static condition in the same range.

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Additionally, 36% of static culture nuclei were found 0-35 o , unlike the stretched condition with 3 6 2 17.6% nuclei in the same range. (Fig. 4B; left panel). In iMSC SCX+ after 7 days, 42.5% of 3 6 3 stretched nuclei displayed an angle of 65-90 o , as opposed to only 26.5% of the nuclei of the static 3 6 4 culture ( Fig. 4B; right panel). In contrast, 30.2% of the static culture nuclei displayed an angle of 3 6 5 0-20 o , while only 15.1% in the stretched plate were found in the same angular range (Fig. 4D).

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Furthermore, the frequency distribution of nuclear angles was skewed towards 90 o , as opposed to 3 6 7 the static culture, which showed a stochastic alignment of nuclei in all directions. Actin filaments 3 6 8 displayed a different orientation distribution in the static culture versus the stretched condition in 3 6 9 both iMSCs and iMSC SCX after 7 days of cyclic stretching. In iMSCs that were stretched, 54.1% 3 7 0 of actin fibers were found to be aligned in 60-90 o angles compared to the axis of stretch, 3 7 1 compared to 33.5% of the static condition. Similarly, in iMSC SCX+ in the stretched condition, 3 7 2 >50% (50.8%) of fibers were found to be aligned in 60-90 o , while in the static condition, there 3 7 3 were similar fractions of fibers aligned in each directionality cluster. For both cell types, even 3 7 4 though the two medians were not statistically different, the frequency distributions between the 3 7 5 two conditions were shown to be significantly different. Cell density was similar for both 3 7 6 conditions and cell types (Fig. 4D). Stretched iMSCs resulted in significantly lower aspect ratios 3 7 7 (Fig. 4E). The frequency distribution of the nuclei in iMSC SCX+ showed that in both conditions, 3 7 8 76% of all nuclei had aspect ratios between 0.55-0.8 (data not shown) but the distributions or 3 7 9 medians were not significantly different (Fig. 4E). 3 8 0

Fig. 4 Cyclic stretching altered nuclear orientation and fiber alignment in iMSCs and iMSC SCX+ . iMSCs and iMSC SCX+ were seeded on deformable silicone plates, stretched in a 2D bioreactor for 7 days ("stretched" condition), fixed with phalloidin for actin filaments (red) and
counterstained with DAPI to mark nuclei (blue). Cells were also plated on similar plates that were placed in the incubator for the same period (static culture controls, "static").

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Further qualitative phalloidin staining of actin fibers and ICC against Tnmd and DAPI in iMSCs 3 8 2 and iMSC SCX+ was performed (Fig.5). Following 7d of uniaxial stretching, quantitative 3 8 3 assessment of median Tnmd intensity showed no difference between any of the groups for both 3 8 4 cell types (data not shown). However, Tnmd staining appeared to be brighter and concentrated 3 8 5 around nuclei in the stretched condition. In contrast, the static culture Tnmd staining displayed a 3 8 6 more diffuse pattern for iMSCs and iMSC SCX+ . 3 8 7 3 8 8 Finally, Collagen-1 staining was performed for iMSCs and iMSC SCX+ following 7d of cyclic 3 8 9 loading and compared to the static condition (Fig. 6). For both cell types, Collagen-1 fiber 3 9 0 deposition seemed to be in parallel to the actin fibers, unlike the static conditions, where the 3 9 1 matrix displayed a more random pattern. Cells were seeded into silicone deformable plates with the same density. In the stretched condition, they were stretched uniaxially for 7 days in a 2D bioreactor, while for the static condition they were placed in the incubator for the same timeframe. Channels are shown individually and merged for the same field of view. A merged higher magnification image is also shown on the right for each cell type and condition.
Dotted line displays the long axis of the silicone plate which was the axis of the longitudinal stretch that was applied with this bioreactor system. Two different magnifications are shown for the two conditions. Scale bars represent 130μm (yellow) and 60μm (white). and iMSC SCX+ (B) were seeded into silicone deformable plates with the same density. In the stretched condition, they were stretched uniaxially for 7 days in a 2D bioreactor, while in the static they were placed in the incubator for the same timeframe. Channels are shown individually and merged for the same field of view. A merged higher magnification image is also shown on the right for each cell type and condition. Dotted line displays the long axis of the silicone plate which was the axis of the longitudinal stretch that was applied with this bioreactor system. Two different magnifications are shown for the two conditions. Scale bars represent 130μm (yellow) and 60μm (white). In the present study we investigated the effect of combined stable Scx overexpression and 3 9 7 biomechanical stimulation to induce tenogenic differentiation of BM-MSCs and iMSCs. We 3 9 8 demonstrated the following: (a) lentiviral vector-mediated stable overexpression induced 3 9 9 tenogenic differentiation in vitro (Fig. 1); (b) when altering substrate stiffness, cell culture on 4 0 0 softer substrates did not promote tenogenic differentiation of BM-MSCs or iMSCs compared to 4 0 1 standard tissue culture polystyrene with and without Scx overexpression (Fig. 2); (c) uniaxial 4 0 2 cyclic stretching was able to induce upregulation of tenogenic markers in both iMSCs and 4 0 3 iMSC SCX+ at day 7, with significantly higher levels of expression in the iMSC SCX+ group (Fig. 3); 4 0 4 (d) uniaxial stretching resulted in changes in nuclear orientation and cytoskeleton alignment in 4 0 5 iMSCs and iMSC SCX+ cells (Fig. 4); and (e) Scx overexpression in iMSCs combined with 4 0 6 stretching resulted in Collagen-1 fiber alignment and Tnmd deposition, indicating 4 0 7 complementing effects of biological and mechanical cues to induce tenogenic differentiation in 4 0 8 iMSC SCX+ compared to iMSCs (Fig. 5-6). To our knowledge, this is the first study to that Scx is an important transcription factor that regulates syndetome specification and tendon 4 1 5 formation. 34; 47 Specifically, we demonstrated that BM-MSC SCX+ cells significantly upregulated 4 1 6 Bgn, Thbs4, and Dcn after 12 days of culture compared to their own baseline. Compared to naïve 4 1 7 BM-MSCs, Thbs4 was increased and Tnmd decreased in BM-MSC SCX+ cells (Fig. 1C). In