Brachyury protein

Frozen vials were thawed in a 37°C water bath for 2 min 30 s. Thawed cells were immediately diluted dropwise using TeSR-E8 medium, plated onto vitronectin-coated culture vessels without washing and placed into a 37°C 5% CO₂ incubator. The split ratio at thawing was 1:1 in the DE algorithm experiments and 1:6 otherwise. Twenty-four hours later, the post-thaw culture was washed with PBS containing Ca²⁺ and Mg²⁺ and stained for esterase activity using calcein AM (Invitrogen). Fluorescence intensity of the stained cells were measured using a Synergy HT microplate reader (BioTek) with an ex/em 485/528 nm filter set. Cells cryopreserved using DMSO and plated post-thaw were used as a control. The ratio of fluorescence measurement of each DMSO-free sample to that of the DMSO control (or post-thaw reattachment rate) was used as the metric of optimization. In addition, fresh cells with the same cell count as cryopreserved cells pre-freeze were used as a positive control, passaged at the same split ratio and stained with calcein AM 24 h post-passage.
Growth in post-thaw culture of cells cryopreserved in the optimized DMSO-free solution was assessed label-free by imaging the culture daily with a Cytation 1 cell imaging multi-mode reader (BioTek) with a 4× objective (NA 0.13, Olympus) in the bright-field and scan mode using default focusing method. Images were automatically analyzed by the Gen 5 software (BioTek) using boundary recognition to measure the confluence. On day 4 after thawing as a result of three stress cycles of freezing, thawing and culturing for one passage, the cell colonies were examined for protein expression by immunocytochemistry using antibodies to detect Nanog (R&D Systems) and Oct4 (Millipore), counterstained with Hoechst 33342 (Invitrogen) and quantified using FIJI (ImageJ). Cells dissociated from the post-thaw culture were examined with anti-Tra-1-60 antibody and its isotype control (Invitrogen) and measured using a BD Accuri C6 flow cytometer (BD Biosciences). Also on day 4, the post-thaw culture was differentiated into all three germ layers using a STEMdiff trilineage differentiation kit (STEMCELL Technologies) over 7 days. The endoderm was detected with anti-FOXA2 and anti-SOX17 antibodies (R&D Systems), the mesoderm with anti-BRACHYURY (R&D Systems) and anti-HAND1 (Invitrogen) antibodies, the ectoderm with anti-PAX6 (DSHB) and anti-NESTIN (STEMCELL Technologies) antibodies, and all counterstained with DAPI (Roche). Fluorescent secondary antibodies (Invitrogen) were utilized to detect primary antibody binding and imaged using the Cytation 1 cell imaging multi-mode reader with a 20× objective (NA 0.45, Olympus).

Rats were euthanized via excess carbon dioxide inhalation, and the coccygeal discs were collected 6 weeks after implantation, and an immunohistochemical analysis was performed for aggrecan and type II collagen. We performed immunofluorescence analysis for a calcitonin gene receptor protein (CGRP), Tie2, brachyury, matrix metalloproteinase-13 (MMP-13), human nuclei antibody, tumor necrosis factor-alpha (TNF-α), and interleukin-1-beta (IL-1β). The harvested tissues were fixed overnight in a 4% paraformaldehyde (PFA) solution and decalcified by using a decalcification solution (RapidCal Immuno; BBC Biochemical, Mount Vernon, WA, USA) for 2 weeks. Then, discs were embedded within paraffin wax and sectioned longitudinally using a microtome (Leica) into sections of 5–10 µm thickness. For the immuno-staining, the first sections were dewaxed, rehydrated, and after that stained with primary antibodies against aggrecan (ab36861; Abcam, Cambridge, UK, 1:1000), type II collagen (ab34712; Abcam, Cambridge, UK, 1:100), CGRP (ab47027; Abcam, Cambridge, UK, 1:200), Tie2 (NBP2-20636; Novus Biologicals, Littleton, CO, USA, 1:200), brachyury (sc-166962; Santa Cruz, Dallas, TX, USA, 1:200), human nuclei antibody (MAB1281; Sigma Aldrich, St. Louis, MO, USA, 1:200), TNF-α (ab6671; Abcam, Cambridge, UK, 1:200), MMP-13 (ab39012; Abcam, Cambridge, UK, 1:200), and IL-1β (AF-501-NA; Novus Biologicals, Littleton, CO, USA, 1:200). Then, after 24 h of incubation, sections were washed with phosphate-buffered saline with Tween 20 and again incubated with the secondary antibody anti-Rb horseradish peroxidase (Roche Diagnostics Ltd., Basel, Switzerland), and Alexa Fluor 488 (A11034; Invitrogen, Waltham, MA, USA, 1:400), Alexa Fluor 488 (A11029; Invitrogen, Waltham, MA, USA, 1:400), Alexa Fluor 568 (A10042; Invitrogen, Waltham, MA, USA, 1:400), and Alexa Fluor 647 (A21469; Invitrogen, Waltham, MA, USA, 1:400)-conjugated secondary antibodies. After that, specimens were carried out for the washing step, then the specimens were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (Abcam, Cambridge, UK, 1:1000) and incubated for 10 min. The sections were mounted and finally examined using a fluorescence microscope (Zeiss 880, Oberkochen, Germany, and Leica SP5, Wetzlar, Germany). The percentages of the positive area for aggrecan and type II collagen and positive cell number relative to DAPI for CGRP, Tie 2, brachyury, MMP-13, human nuclei antibody, TNF-α, and IL-1β were calculated using ImageJ software (Version 1.50b, https://imagej.nih.gov/ij/ (accessed on 10 November 2022)).

The STEMdiff Endothelial Differentiation Kit (STEMCELL Technologies) was used according to the manufacturer’s instructions for the differentiation of iPSCs into eMPCs and ECs. Initially, iPSCs were detached with StemPro Accutase (Thermo Fisher Scientific) and seeded at a density of 100,000 cells per 6-well in Essential 8 Flex medium supplemented with 10 µM ROCK inhibitor Y-27632 (STEMCELL Technologies). On day 7, differentiated ECs were subcultured at a density of 150,000 cells per 6-well. The cells were expanded in STEMdiff Endothelial Expansion Medium (STEMCELL Technologies) for four days, then split on passage #1, and cultured for an additional four days. RNA was extracted for eMPCs on day 3 of the differentiation protocol and at passage #1 for expanded differentiated ECs.
The PSC 4-Marker Immunocytochemistry Kit (Thermo Fisher Scientific) was used to analyze SSEA4, OCT4, SOX2, and TRA-1-60 expression for iPSCs. An anti-mouse Alexa Fluor 555 antibody (ab150114, 1:500, Abcam, Cambridge, UK) and an anti-rat Alexa Fluor 555 antibody (A-21434, 1:500, Thermo Fisher Scientific) were used as alternative secondary antibodies for SSEA4 and SOX2 staining. Unless otherwise specified, the Immunofluorescence Application Solutions Kit (Cell Signaling, Danvers, MA, USA) was used according to the manufacturer’s instructions for marker staining after the differentiation of iPSCs into eMPCs and ECs. For eMPCs, immunofluorescence analysis for α-SMA (ab7817, 1:100, Abcam) and Brachyury (AF2085, 1:20, Bio-Techne, Minneapolis, MN, USA) was performed. For Brachyury staining, cells were permeabilized and blocked in 1% bovine serum albumin, 0.3% Triton X-100, and 10% normal donkey serum. For ECs, endothelial marker expression at passage #1 was evaluated by the staining of CD31 (3528S, 1:800, Cell Signaling), VE-Cadherin (2500S, 1:400, Cell Signaling), and VWF (MA5-14029, 1:66, Thermo Fisher Scientific). An anti-mouse Alexa Fluor 555 antibody (ab150114, 1:200, Abcam), an anti-rabbit Alexa Fluor 555 antibody (A-21429, 1:500, Thermo Fisher Scientific), and an anti-goat Alexa Fluor 555 antibody (ab150130, 1:200, Abcam) were used as secondary antibodies. DAPI or Hoechst 33342 (Thermo Fisher Scientific) were used for nuclei staining.

PCR was performed with the Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). DNA was purified using a GeneJET Gel Extraction Kit (Thermofisher, Scientific, Waltham, MA, USA). All DNA Fragments were ligated by the One Step Cloning Kit (Vazyme, Nanjing, China).
To identify the transcriptional activation activity, the isoform I, isoform II, and truncated isoform I of CrDYRK1 were amplified from C. robusta cDNA separately using PCR and ligated to pGBTK7 (BD) vector (Takara, Beijing, China), respectively. The HsDYRK1A (residues 485–763) and HsDYRK1B (residues 437–629) fragments were amplified separately from human cDNA by PCR and ligated to BD vector for the transcriptional activation activity analysis. The CsDYRK1 (residues 393–626) fragment was amplified from C. savignyi cDNA by PCR and ligated to BD vector for the transcriptional activation activity analysis. The DmMNB (residues 629–1047) fragment was amplified from Drosophila melanogaster cDNA by PCR and ligated to BD vector for the transcriptional activation activity analysis.
In the repression assay, CrNFAT5 (residues 77–979) and CrMyc (residues 1–681) fragments were amplified and ligated to BD vector to serve as a positive control. CrDYRK1 (isoform II) (residues 1–168) fragment was amplified and subcloned into EcoRI restriction sites of the CrNFAT5 (residues 77–979)-BD vector. CrDYRK1 (isoform II) (residues 1–168) and CrMyc (residues 1–681) fragments were amplified separately and ligated to BD vector to construct an expression vector CrDYRK1 (isoform II) (residues 1–168)-CrMyc (residues 1–681)-BD. Truncated versions of CrDYRK1 (isoform II) were amplified separately and ligated to BD vector to construct expression vector CrDYRK1 (isoform II) (residues 43–815)-BD and CrDYRK1 (isoform II) (residues 85–815)-BD. HsDYRK1A-N (1–69) fragment was amplified and subcloned into EcoRI restriction sites of the HsDYRK1A (residues 485–763)-BD vector to construct expression vector HsDYRK1A-N (residues 1–69)-C (residues 485–763)-BD.
In the RNA-seq assay, HsDYRK1A (residues 485–763) fragment was amplified and ligated to pEGFP-N1 vector (Addgene, Watertown, MA, USA). HsDYRK1A-N (1–69)-C (485–763) was amplified from HsDYRK1A-N (residues 1–69)-C (residues 485–763)-BD vector and ligated to pEGFP-N1 vector.
In the assay of subcellular localization observation, the isoform I and isoform II of CrDYRK1 were amplified and ligated to pEGFP-C1 vector driven by Brachyury promoter. HsDYRK1A (residues 485–763) fragment was amplified by PCR and ligated to pEGFP-N1 vector. CsDYRK1 (residues 393–626) and CrDYRK1 (isoform I) (residues 427–647) fragments were amplified by PCR and ligated to pEGFP-N1 vector driven by Epi I promoter, respectively.
All primers are listed in Supplementary Table S1.