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Fractal heterogeneity in minimal matrix models of scars modulates stiff-niche stem-cell responses via the nuclear exit of a mechanorepressor

Scarring is a long-lasting problem in higher animals, and reductionist approaches could aid in developing treatments. Here, we show that co-polymerization of collagen-I with polyacrylamide produces minimal matrix models of scars (MMMS), in which fractal-fiber bundles segregate heterogeneously to the hydrogel subsurface. Matrix stiffens locally – as in scars – while allowing separate control over adhesive-ligand density. The MMMS elicits scar-like phenotypes from mesenchymal stem cells (MSCs): cells spread and polarize quickly, increasing nucleoskeletal lamin-A yet expressing the ‘scar marker’, smooth muscle actin (SMA) more slowly. Surprisingly, expression responses to MMMS exhibit less cell-to-cell noise than homogeneously stiff gels. Such differences from bulk-average responses arise because a strong SMA repressor, NKX2.5, slowly exits the nucleus on rigid matrices. NKX2.5 overexpression overrides rigid phenotypes, inhibiting SMA and cell spreading, while cytoplasm-localized NKX2.5 mutants degrade in well-spread cells. MSCs thus form a ‘mechanical memory’ of rigidity by progressively suppressing NKX2.5, thereby elevating SMA in a scar-like state.


Estimation of MMMS collagen concentration
From confocal stack reconstruction, we found that embedded collagen is enriched within 10 microns of the upper surface of MMMS gels of ~80 micron thickness ( Supplementary Fig. S2a). For MMMS gels, surface coverage of collagen fiber bundles is ~30% based on Sirius red and/or collagen immunofluorescence staining, which matches that of mdx and DMD skeletal muscle tissue cross-sections (~20-30%; refs. 1 and 2). This leads to a corrected collagen concentration from the nominal value of 0.4 mg/mL to an effective c ~ 12 mg/mL. Bulk measurements of scarred mdx muscle yield 2-3 mg/ml hydroxyproline 3 , which is 10-20% of collagen mass, so that such scarred tissues have a mean collagen concentration of 10-30 mg/mL, which is very similar to our estimate for the MMMS system.

Adhesion and Thin-film Corrections for MMMS gel
In order to create 2D hydrogel substrates for cell culture, polyacrylamide (PA) gels were sandwiched between an allysilanated bottom coverslip and a nonreactive top coverslip (18 and 25 mm in diameter, respectively; Fisher). Embedding fibrous collagen in polyacrylamide hydrogels exhibited tackiness to the gel surface post-polymerization. That is, removal of the top coverslip required more force than without the EC fibers. This again provided evidence for interfacial localization of the fibers. To correct for tackiness of MMMS gel, the force required to remove the top coverslip was measured with a custom-made and calibrated polystyrene cantilever (spring constant k sp = 804.6 N/m), attached at one end to a syringe pump (RS-232, Harvard Apparatus) and the other to a nylon string, which in turn was firmly attached to the top coverslip of a stage-immobilized gel. Syringe pump was set to 'pump mode' pulling the cantilever-string-coverslip setup at an effective velocity of 0.4 mm/s. The process of removing the top coverslip was observed with a side-view camera to determine when the pulling string should be stopped and maintained at constant force (zero velocity). Maximal deflection of the cantilever (translated to maximal force of the pulling string) was observed before coverslip was fully detached (or before cantilever relaxes back to zero deflection) and was used to calculate gel adhesion force. Adhesion force on MMMS gels was found to be 84 ± 20% higher than on pristine 0.3 kPa gels (n = 5).
The effective elasticity can be derived from rigid cylinder (i.e. coverslip of length a) pulling from a soft elastic thin film by assuming non-uniform Boussinesq-type stress distribution at the interface. The force per unit length F/a (along x) or P that propagates from the edge of the coverslip can be derived as 4 : where E is Young's modulus of the medium, σ is Poisson's ratio, and = ! + ! . A similar formula for ! was also found. We assumed that the lateral pulling force was applied to a small region and that the measured displacement profiles were obtained at large distances away from the initial loading point to satisfy Equation (8). In the simplest case where x = r (y = 0), displacement far enough from the loading force (Boussinesq approximation) scales as ! = ! / , for some Boussinesq constant, ! . Since such scaling leads to infinite displacement at x = r = 0, we also assumed exponential scaling ( ! = ! !!" , for some characteristic decay constant b; Fig. 1E, iii) to set the observed maximum displacement, ! , at the initial point. Both scalings agree well (R 2 > 0.9), as evidenced by the fitted scaling constants ( Supplementary Fig. S4e).

Mechanobiological Gene Circuit (MGC) Model for Matrix-directed Osteogenesis
Lamin-A (L) and smooth muscle actin (S) message and protein circuitry is schematically presented in Supplementary   Fig. S11c. Expression kinetics is described by a set of coupled rate equations for the respective transcripts (lower case) and proteins (upper case), as follows: !" !" In this parsimonious model, RNA degradation and translation were assumed linear in transcript concentration. Lamin-A protein positively regulates one of its transcription factors, RARG, and smooth muscle actin (SMA) protein positively regulates one of its transcription factors, SRF, so that each enhances its own transcription (with rate constants α ! , α ! ). In addition, Lamin-A protein also enhances SMA transcription via the SRF pathway (rate constant α ! ). To model stress-stabilized protein assembly, we describe lamin-A and myosin-IIA protein turnover with suitable Hill models (rate constants δ ! , δ ! ).
Specifically, Lamin-A protein turnover is suppressed by SMA-generated stress with K ! = S ! ! , while SMA protein turnover depends on matrix elasticity E: to collectively obtain best agreement with experimental data (Supplementary Fig. S8a). In particular, cooperative protein turnover was n ! = 10.4 and n ! = 2 with the half-maximum effect on SMA turnover occurring at E ! = 5 kPa. All other parameters were of order of magnitude 1. As matrix stiffness and cell tension suppresses protein phosphorylation and turnover 8,9 , steady-state levels monotonically increase with matrix E, consistent with the coupled mechano-regulation of lamin-A and SMA ( Supplementary Fig.   S8a).

Supplementary Outlook
Displacement profiles on stiff fiber bundles on MMMS (Fig. 1e) help answer how far away does a cell feel a scar. The profiles show that a cell 10 mm away can sense a stiff circular island of 30-mm radius, so that the effective 'sensed area' increases from ~3000 sq. mm to 5000 sq. mm. Thus, the 20-30% area coverage by these islands (Supplementary Fig. S6) effectively increases to ~30-50%, notwithstanding that these stiff 'islands' have a fractal, rather than round, shape.
MSC plasticity was also exploited to mimic and clarify fibrosis mechanisms in vivo. MSCs recapitulated the expression changes detectable in scarred striated muscle (Fig. 1b,c). Our reductionist approach in vitro to scarred tissues by using MMMS helped clarify cell responses in both short and long time-scales. That a heterogeneous MMMS elicits less cell-cell heterogeneity than homogeneous substrates ( Fig. 3 and Supplementary Fig. S8) highlights the importance of ECM micro-architecture in the niche and mechanochemical control of cell fate. Such a finding is difficult to explore with homogeneous and isotropic substrates alone. The findings also underscore the relative contributions of cell-intrinsic versus extrinsic variation to cell-material interactions and cell biology in general. Single-cell analyses use many types of new and sensitive detection and sequencing technologies, but they do so after plucking cells from cultures or poorly understood tissues; tissue-relevant insights can thus be obtained if matrices with bioinspired fractal heterogeneity are used rather than homogeneous substrates. For example, a better model of cancer-associated fibrosis 10 (e.g. Fig. 1g) could be helpful for understanding such matrix remodeling and heterogeneity as a major risk factor for cancer (e.g. liver cancer) that likely affects a cancer stem cell in its niche as well as a tissue stem cell in its niche. Single-cell analyses of such important processes and cell types may be artificially limited when using homogeneous substrates that broaden phenotypic variation.
The kinetics of various mechanosensitive proteins showed that MSCs responded to our heterogeneous MMMS gels as if they were on much stiffer, homogeneous matrices -which is surprising in that it requires MSCs to 'remember' stiff domains more so than soft domains of a matrix. Myosin-II polarization is implicated in the short-term mechanical memory imparted by the rigid regions of MMMS.  Table S3), which could impede pre-mRNA processing and transport as already documented for actin 15,16 . Fourth, Nkx2.5 binds to the lamina-associated polypeptide-2 (LAP2, or TMPO) (Supplementary Table S3), which promotes differentiation of muscle stem cells 17 , so that Nkx2.5 could contribute to muscle injury processes (Fig. 1c) and also crosstalk with the lamin-A gene circuit. A multiplicity of such nuclear interactions of Nkx2.5 8 dimers 18 is further consistent with the cooperative nature of SMA de-repression on stiff matrix ( Fig. 4b fits have Hill exponents >2): the more NKX2.5 is in the nucleus, the more it is retained to repress SMA.
SMA-high cells (strong Responders) on homogeneously stiff substrates are thus more sensitive to minor changes in nuclear-localized NKX2.5, compared to weak Responders (Fig. 4b, right inset and Supplementary Fig. S9). Such sensitivity equates to the derivative, ∂[SMA] / ∂[NKX2.5 nuclear ] and helps explain why the heterogeneity of cells on homogeneously stiff matrices is greater than cells on MMMS and CC 0.3kPa gels (Fig. 3). For MMMS importantly, a cell on a fractal fiber island that upregulates SMA and downregulates nuclear NKX2.5, will later migrate off of this stiff island ( Supplementary Fig. S5) and potentially reverse the process in a way that cannot happen on a homogeneous substrate.
The many nuclear interactions of Nkx2.5 combined with its strong NLS (Figs. 4f, 5) would tend to oppose nuclear export and cytoplasmic degradation of Nkx2.5, thus favoring its nuclear import and retention ( Supplementary Fig. S12). Histones also have multiple DNA and protein interactions that strengthen nuclear retention and impede turnover: every one of the 18 histones detected in a recent proteomics study of protein turnover in fibroblasts cultured on rigid glass shows a longer half-life than the median (2.0 days for 5028 detected proteins, with histone half-lives ranging from 2.4 to 8.7 days 19 ).
Turnover that takes many days fits our NKX2.5 kinetics for MSCs on rigid matrix (Fig. 4b). Importantly on rigid matrix (but not on soft matrix), some of the nuclear interactions of NKX2.5 could weaken and slowly tilt the balance away from nuclear retention. In particular, NKX2.5 could be displaced from chromatin binding by rigidity-driven accumulation of nuclear factors in the SRF pathway, which coregulates ACTA2 expression 20 and is coupled to the lamin-A gene circuit 9 . Interactions with nucleusretained proteins alone may not sufficiently explain the slow NKX2.5 translocation, and regulation by other mechanotransduction pathways requires exploration. Ultimately, extensive mutagenesis beyond our in-depth analyses of the NLS domain in NKX2.5 might one day map each of NKX2.5's many interaction partners and thereby deepen insight into mechanisms and additional pathways responsible for our observation of a slow, rigidity-driven decrease in NKX2.5's nuclear localization. Contributions of individual amino acids to how MSCs gradually acquire long-term mechanical memory might then become clear.
MSCs are more multipotent than lineage-restricted fibroblasts 21 , but SMA expression in fibroblasts is well documented in fibrosis (e.g. ref. 13 ). Moreover, single cell profiling of mouse fibroblasts recently implicated a role in scarring for the cell surface peptidase Cd26/Dpp4, which -when inhibited by a small molecule -diminished scarring 22 , and we find CD26 in human MSCs decreases with overexpression of NKX2.5 as well as with knockdown of LMNA ( Supplementary Fig. S12). While we have shown MMMS 9 decreases nuclear NKX2.5 and increases LMNA in MSCs, any changes in additional fibroblast proteins (e.g. CD26) due to MMMS require further study. More broadly, the fact that scarring occurs progressively only in mature stages of higher animals 23 likely reflects an evolved need to generate a sufficiently stiff tissue that sustains large stresses in mature tissues. Tissue injuries initiate mechanical as well as biochemical changes that can serve as homing cues for highly plastic MSCs. For stem-cell based therapies in which differentiation-competent cells are injected into a wound, our findings provide a guide to controlling cell responses to scars that range from contractile induction to ectopic calcification 24 .

Proteomics Analyses
Mouse tissue was prepared for analysis as described by Swift et al. 9 . Briefly, approx. 1 mm 3 of frozen muscle tissue was ground on dry ice and solubilized in 100 µL 1x NuPAGE LDS sample buffer (Life Technologies); samples were sonicated on ice before addition of 1% β-  The IP samples were prepared for MS analyses as detailed in ref. 25  Proteins pulled down by both control rabbit IgG and rabbit anti-NKX2.5 IgG were not included from the final list of proteins (Table S3).

Bulk Rheology
A cone-and-plate Bohlin rheometer (Malvern Instruments, Worcestershire, UK) was used to measure the viscoelastic spectrum (elastic modulus, G', and viscous modulus, G") of the hydrogels as a function of frequency. In order to confirm polymerization had come to completion, a time sweep test was also performed for 20 minutes with a controlled absolute strain of 1% at an angular frequency of 1 rad/s. PBS was then added to allow complete swelling of the gel before a frequency test was conducted from 25 rad/s down to 0.005 rad/s at 1.0% strain.

Fractal Dimension Analysis
Fractal dimensions were obtained from immunofluorescence images of MMMS and collagen-coated PA gels (and tissue culture plastic) to quantify collagen architecture ( Supplementary Fig. S2b), as recently suggested 26 . We used a fractal box-count method using ImageJ software: images were transformed into binary and a built-in fractal box count plugin was used to obtain fractal dimension values. The range of fractal dimension values (within the limits of 1.0 -2.0) was confirmed by processing a blank image and a filled image (e.g. tissue culture plastic), respectively.
Collagen-coated gels exhibit fractal dimensions that are similar to a filled image, while increasingly fractal-like images approach the midpoint (~1.5).

3D reconstruction and Photobleaching in Confocal Microscopy
To visualize both PA gel and collagen-1 (Fig. 1D), the gel precursor was doped with 0.02% w/v allylamine (Sigma) for subsequent fluorescein isothiocyanate (FITC, Thermo Scientific) conjugation, while collagen-1 coating covalently crosslinked with sulfo-SANPAH on the gel surface was indirectly immunolabeled (secondary antibody: donkey anti-mouse Alexa-647 fluorophore, Life Technologies). To delineate registration of collagen coating on polyacrylamide gels, confocal microscopy (FV1000, Olympus) with 40x objective (UPLFLN40XO, N.A. 1.3) was used. Pinhole size was set at 125 µm to generate an optimal slice thickness of 0.76 µm. Collagen coat is ~5 µm on a fully hydrated hydrogel of ~80 µm thickness as determined by 3D confocal stack reconstruction.
Fluorescence photobleaching was also performed to determine the extent of immobilization of the collagen-I coat on the gel surface ( Supplementary Fig. S4d). Gel surface fluorescence was imaged prior to bleaching. Then the confocal plane was localized on the gel surface as determined by collagen-I immunofluorescence, and laser power was increased to maximum possible amount to photobleach a circular region of interest (ROI) for 60 seconds. Gel surface with the photobleached ROI was re-acquired for another 5 minutes. Even without sulfo-SANPAH crosslinking, photobleached collagen-1 remains relatively stable perhaps due to interpenetration into the inert porous gel. instructions. Prior to plating into decellularized heart matrices, MSCs were transduced at passage 2 (50% confluency) with 300 PFU of lentivirus per cell for 24 hrs to stably express GFP.

Chick Embryonic Heart Matrix Decellularization and Cardiomyocyte Isolation
White Leghorn chicken eggs (Charles River Laboratories) were incubated at 37°C, rotated once per day, until the desired developmental stage was reached. Hearts were removed from chicken embryos at embryonic day 14 and the left ventricle wall was isolated and immersed in 1% SDS in deionized water for decellularization.
After shaking overnight at room temperature (RT), the decellularized tissue was placed into 1% Triton-X in deionized water for 30 min and then equilibrated in PBS with 100 ug/mL penicillin and 100 uM streptomycin until used for experiments. To make a gradient of crosslinks in the matrix, the decellularized tissue was fixed on a coverslip and then placed into a chamber such that approximately one half is immersed in 100% glycerol. After allowing glycerol to penetrate into the matrix for 30 min, 3 mM of genipin (a fluorescent crosslinker, Sigma) in PBS was added on top of the glycerol to immerse the other half of the matrix, and was then incubated in the dark at 37°C for 2 hours. Glycerol was then added to overflow the genipin solution, and the resulting matrix with a crosslink gradient was washed and stored in PBS with antibiotics prior to use.
For cardiomyocyte-on-gel cultures, embryos were extracted at room temperature by windowing eggs, removing extraembryonic membranes with forceps and cutting major blood vessels to the embryonic disc tissue to free the embryo. For E2-E5 embryos, whole heart tubes (HTs) were extracted by severing the conotruncus and sino venosus. All tissues were incubated at 37°C in pre-warmed chick heart media (alpha-MEM supplemented with 10% FBS and 1% Penicillin/Streptomycin, Gibco, 12571-063) until ready for use. Cell isolation from heart tissue was performed by dicing sub-millimeter size and then digesting with trypsin/EDTA (Gibco, 25200-072). To digest, we incubated tissue in approximately 1 mL trypsin per E4 HT for 13 min rotating at 37°C, for 2 min upright to let large tissue pieces settle before carefully removing supernatant and replacing with an equal volume of fresh trypsin, and finally shaking for 15 more min. We stop digestion by adding an equal volume of chick heart media. Cells were plated at concentrations of approximately 2 × 10 5 cells/cm 2 directly on collagen-1-coated PA gels of varying stiffness.
Imaging of MSC migration was done using an inverted Olympus IX-71 microscope with a 10x objective enclosed in a humidified chamber at 37°C and 5% CO2, 300 W xenon lamp illumination, and a high-resolution CCD camera (Photometrics CoolSnap HQ). Deltavision Softworx software was used for image acquisition at 10-min intervals. ImageJ was used to track the center of MSC nuclei in movie sequences, and the summed contour distance traveled divided by the time was used as our measurement of mean speed. This measurement only reflects total distance traversed but does not reflect cell persistence. We quantified the bias of the time-dependent number of steps to the left (L, soft CC 0.3kPa region) and to the right (R, MMMS region) by using the equation adapted from Isenberg et al. 27 : Durotaxis Index = (R -L) / (R + L). If Durotaxis Index = 0, there is no bias to cell migration. If Durotaxis Index = 1, all of the steps are toward the stiff side. Cells that underwent mitosis and those that migrated out of the trackable region were included only prior to such events.

Quantitative Immunofluorescence Microscopy
For immunofluorescence staining, cells were fixed with 3.7% formaldehyde (Sigma) in PBS for 15 min at RT and washed with PBS twice for 5 min.
Blocking was done for 1 hr with 1% BSA in PBS. All primary antibodies (see Supplementary Table S2 for dilutions) in PBS with 0.025% Tween-20 (PBST) were incubated at RT for 1 hour or overnight at

Cell Morphometrics
Intensity analyses and cell-morphology quantification were performed in ImageJ software. From images acquired from immunofluorescent samples, F-actin and myosin IIB rear/front polarization ratios were determined by splitting the cell into two equal areas (rear and front). The ratio is the total integrated fluorescence of the rear half over the front half of the cell. Cell area and aspect ratio were quantified from myosin IIA or F-actin immunofluorescence (since they consistently stain the whole cell body). Before comparing different sample conditions, integrated intensity of a particular protein was normalized to background value (secondary antibody only control).

Transcriptional profiling by DNA microarrays
Total RNA was extracted from cells using Trizol and purified by RNeasy (Qiagen) with on-column DNase digestion according to manufacturer's protocol.
Adherent cells were gently scraped in Trizol. Total RNA was amplified and converted to cDNA using WT-Ovation Pico kit (NuGen). Fragmented and biotin-labeled ST-cDNA was generated using WT-         repair of injured tissue such as an infarcted heart are controversial 32 , but evidence for cell trafficking from soft marrow to an injured site such as a stiff scar is compelling 11 . Such migration will typically involve a large gradient in stiffness (such as an infarct border zone of ~9 kPa/mm 33,34 ), and with several other homogeneous gel systems, various cell types including MSCs have been seen to 'durotax' from soft to stiff matrix 8,27,33,35 . Here, a gradient of increasing embedded-collagen fiber bundles was made to resemble a transition from homogeneous soft tissue to a heterogeneous scar. Importantly, matrix heterogeneity did not obscure durotaxis probably because the turnover of cytoskeletal structures (e.g. MIIB) takes hours 8 . Cells thus average over fractal heterogeneities and 'remember' only the stiffer fiber bundles -which is not addressable with isotropic substrates that are of unknown or questionable relevance to scars. Haptotaxis did not contribute because MSCs did not spread on MMMS without the additional collagen coating (Supplementary Fig. S3a). Moreover, the minimum area fraction of 'scar' for imparting a mechanical memory is estimated to be 10-15% since MSCs durotax into an increasingly heterogeneous scar-like matrix, with migration results similar to those on homogeneous gels.

Figure S9
MSCs at day 2 respond to MMMS and stiff matrix by

Figure S11 | Stiffness of MMMS increases lamin-A and osteo-commitment of MSCs.
Our analyses of striated muscle diseases prone to scarring revealed an increase in lamin-A (Fig. 1C, S1), which is a nucleoskeletal protein implicated in various differentiation and maturation processes 9, 38, 39 . a, Lamin-A regulates nuclear entry of a retinoic acid (RA) receptor (RARG), which is highly expressed in bone 9 , and osteogenesis is also positively regulated by the SRF pathway 40 , which appears stiffness-activated.
Previous studies further showed that MSCs injected into infarcted hearts can sometimes form calcified structures 24 that would be detrimental to the beating heart. Given the long-term maintenance of a scar-   consensus sites for NKX2.5 (ref. 13 ) and SRF (refs. 43 ) are also shown. Two NKX2.5 binding sites have been shown to synergistically repress ACTA2 13 , possibly in a dimerized form 18 . This repressive loop is reminiscent of the well-studied LacI repressor: when a series of repressive loops are upstream of an induced promoter in interphase cells, transcription doubles by half-hour after induction as the chromatin visibly de-condenses 44 . In other words, de-condensation does not occur in seconds. For the NKX2.5 system here, a repressive-dimer loop may need to be lost before SRF can drive ACTA2 transcription, and this de-condensation could take 1-2 orders of magnitude longer. Right, More importantly, multiple nuclear interactions (Table S3A)