Force and scleraxis synergistically promote the commitment of human ES cells derived MSCs to tenocytes

Xiao Chen1,2 #; Zi Yin1,2 #; Jia-lin Chen1,2; Wei-liang Shen3; Huan-huan Liu1,2; Qiao-mei Tang1 2; Zhi Fang1 2; Lin-rong Lu 4; Junfeng Ji 1 **; Hong-wei Ouyang1,2,4*

1 Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang University, Hangzhou, China, 310058

2 Zhejiang Provincial Key Laboratory of Tissue Engineering and Regenerative Medicine, School of Medicine, Zhejiang University, Hangzhou, China, 310058

3 Department of Orthopedic Surgery, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China, 310058

4 Institute of Immunology and Program in Molecular and Cellular Biology, Zhejiang University School of Medicine, Hangzhou China 310058

#These authors contributed equally to the study.

Supplementary data

Supplementary Figure 1 scleraxis overexpression. (a) qPCR analysis on SCX gene expression of control and SCX overexpressing cell sheet at 0, 3, 7, 11 and 16 days after reaching confluence. Transcripts were normalized to GAPDH. (b) SCX-GFP transfection showed high expression and nuclear localization of SCX in hESC-MSC. Scale bar, 50um. (c) Western blot analysis of SCX expression on cells infected with SCX-GFP, GFP and SCX. Note that the molecular weights of SCX and SCX-GFP were 22-26KDa and SCX-GFP was 52kDa, respectively. Lane1: SCX-GFP , Lane2: GFP , Lane3: SCX..

Supplementary Figure 2 in vitro construction of engineered tendon. (a) Scaffold-free engineered tendon after mechanical stress. (b) TEM results of engineered tendon formed after 2 weeks showing the collagen fibrils formation under different conditions..

Supplementary Figure 3 Densitometric analysis of pSmad activity .(**, P<0.01)

Supplementary Figure 4 Ectopic engineered tendon in vivo. (a) The survival of grafted hESCs derived cells was detected using anti-human-positive nuclei (anti-HuNu) staining (green). Nuclei are stained by 4', 6-diamidino-2-phenylindole (DAPI). Scale bar = 20um. (b) Noninvasive monitoring of cell survival in the implantation site. Engineered tendon stained with DiI was implanted in dorsal subcutaneous site of nude mice. CCCD analysis demonstrated a positive DiI signal detected in the implantation site (arrow), indicating the survival of implanted cells. (c) Quantitative analysis on collagen diameter of ectopic engineered tendon in vivo under different conditions (*: SCX and mechanical interaction, p<0.05).
Supplementary Table

Table s1 Antibody used in this study

antibody / company / catalog number
Collagen XIV / Abcam, www.abcam.com / ab58084
Collagen I / Abcam, www.abcam.com / ab6308
Eya2 / Sigma, www.sigma-aldrich.com / HPA027204
SCXA / Abcam, www.abcam.com / ab58655
Tenomodulin / Abcam, www.abcam.com / ab81328
Runx2 / Abcam, www.abcam.com / ab76956
Human nuclei / Millipore, www.millipore.com / MAB1281

Table s2 Real-time PCR primer for gene expression

genes / primers
Human Six1 / Forward / GGAGTTATTGTTTTCGGTGTTC
Reverse / TGTCCTGCGGGAGTGGTA
Human scleraxis / Forward / CGAGAACACCCAGCCCAAAC
Reverse / CTCCGAATCGCAGTCTTTCTGTC
Human collagen Ia1 / Forward / ATGGATTCCAGTTCGAGTAGGC
Reverse / CATCGACAGTGACGCTGTAGG
Human collagen Ia2 / Forward / GGGCTCTAATGATGTTGAACTTGT
Reverse / ATGATTGTCTTTCCCCATTCATTT
Human collagen XIV / Forward / AAGGATTGCCCTCCGACTACAC
Reverse / CTGATGCGTTCATTGCCTTCTC
Human collagen III / Forward / TTTTGCAGTGATATGTGATGTT
Reverse / GGATGGTGGTTTTCAGTTTA
Human tenomodolin / Forward / TGGGTGGTCCCTCAAGTGAAAGT
Reverse / CTCGACGGCAGTAAATACAACAATA
Human Epha4 / Forward / TGTGGGCTGTGACAATCTGGAATA
Reverse / CATTTAGACGGAACTGAGGAGGGT
Human Eya2 / Forward / CAACGTTGGTGGGTTGATAGGCA
Reverse / GGCCAGGGCAGGAATTAGTTGAGT
Human OCN / Forward / CGCAGCCACCGAGACACCAT
Reverse / TAGACCGGGCCGTAGAAGC
Human BMP2 / Forward / CGGGAGAAGGAGGAGGCAAAGA
Reverse / GGGAAGCAGCAACGCTAGAAGACA
Human Smad8 / Forward / CACGGCTTTGAAGTCGTGTAT
Reverse / TGAAGAAATGGGGTTATGTGGA
Human GAPDH / Forward / TGACGCTGGGGCTGGCATTG
Reverse / GGCTGGTGGTCCAGGGGTCT


Supplementary methods

Cell labeling and detection. To genetically identify hESC-MSCs within the site of implantation in vivo, the cells were stained with DiI, which is stable in paraffin sections[46]. Briefly, when the cells had reached 80% confluency, DiI was diluted in the culture medium to achieve a final concentration of 10 μg/mL. One million cells were suspended per mL and incubated for 30 min at 37°C. Afterwards, cells were washed twice in PBS. The fluorescence of DiI was measured under fluorescence microscopy (BX41; Olympus, Japan) at an excitation wavelength of 543 nm.

In situ molecular imaging. To evaluate the survival of implanted cells within the tendon defect, a tracking system to detect cell migration was utilized. Since the cells had been stained with DiI before implantation, we were able to detect the fluorescence signal within the implantation site using a tracking system (Kodak MI) consisting of a CCD camera equipped with a 50-mm lens [7].