METHOD FOR INDUCING DIFFERENTIATION OF STEM CELLS

Abstract

A cell culture including a cell culturing medium for growing stem cells, a three-dimensional (3D) cell growth matrix and stem cells, wherein the cell culture has a critical stress .sub.c of 2-30 Pa, wherein the critical stress is a stress which marks an onset of strain stiffening and wherein the cell culture has a storage modulus G measured at 37 C. of 50-1000 Pa.

Claims

1. A cell culture comprising: a) a cell culturing medium for growing stem cells, b) a three-dimensional (3D) cell growth matrix and c) stem cells, wherein the cell culture has a critical stress .sub.c of 2-30 Pa, wherein the critical stress is a stress which marks an onset of strain stiffening and wherein the cell culture has a storage modulus G measured at 37 C. of 50-1000 Pa, preferably .sub.c ranges between 5-25 Pa and G measured at 37 C. ranges between 70-400 Pa.

2. The cell culture according to claim 1, wherein the 3D cell growth matrix comprises at least one of Matrigel, Puramatrix, Raft 3D, Insphero, Bioactive 3D, Cellusponge, Optimaix and GroCell-3D scaffolds.

3. The cell culture according to claim 1, wherein the 3D cell growth matrix comprises an oligo(alkylene glycol) substituted co-polyisocyanopeptide.

4. The cell culture according to claim 3, wherein a concentration of the polyisocyanopeptide in the 3D cell growth matrix is 1-5 mg/ml.

5. The cell culture according to claim 3, wherein an average length of the polyisocyanopeptide is 250-680 nm as determined by AFM.

6. The cell culture according to claim 3, wherein the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide and/or the cell culturing medium comprises fibrin, wherein when the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide, the average distance between the cell adhesion factors along the polyisocyanopeptide backbone is 10-50 nm.

7. A method for inducing differentiation of stem cells, comprising the steps of: a) mixing a cell culturing medium for differentiation of stem cells with an oligo(alkylene glycol) substituted co-polyisocyanopeptide at a temperature between 0 and 18 C. to obtain a polymer solution; b) mixing the polymer solution with stem cells at a temperature between 0 and 18 C. to obtain a cell culture solution; c) allowing the cell culture solution to warm to a temperature between 30 and 38 C. to form a cell culture comprising a hydrogel and allow the stem cells to differentiate, wherein a concentration of the polyisocyanopeptide in the polymer solution is 1-5 mg/ml, wherein an average length of the polyisocyanopeptide is 250-680 nm as determined by AFM, wherein a cell density of the stem cells in the cell culture solution is 0.3*10.sup.6-1*10.sup.6 cells/ml, wherein the hydrogel has a critical stress .sub.c of 2-30 Pa, wherein the critical stress is a stress which marks an onset of strain stiffening, wherein the hydrogel has a storage modulus G measured at 37 C. of 50-1000 Pa and wherein the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide and/or wherein the cell culturing medium comprises fibrin, wherein when the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide, the average distance between the cell adhesion factors along the polyisocyanopeptide backbone is 10-50 nm.

8. A method for inducing osteogenic differentiation of stem cells, comprising the steps of: a) mixing a cell culturing medium for osteogenic differentiation with an oligo(alkylene glycol) substituted co-polyisocyanopeptide at a temperature between 0 and 18 C. to obtain a polymer solution; b) mixing the polymer solution with stem cells at a temperature between 0 and 18 C. to obtain a cell culture solution; c) allowing the cell culture solution to warm to a temperature between 30 and 38 C. to form a cell culture comprising a hydrogel and allow the stem cells to differentiate, wherein a concentration of the polyisocyanopeptide in the polymer solution is 1-5 mg/ml, wherein an average length of the polyisocyanopeptide is 250-680 nm as determined by AFM, wherein a cell density of the stem cells in the cell culture solution is 0.3*10.sup.6-1*10.sup.6 cells/ml, wherein the hydrogel has a critical stress .sub.c of 13-30 Pa, wherein the critical stress is a stress which marks an onset of strain stiffening, wherein the hydrogel has a storage modulus G measured at 37 C. of 50-1000 Pa, preferably between 70-350 Pa, more preferably between 72-300 Pa, and wherein the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide and/or wherein the cell culturing medium comprises fibrin, wherein when the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide, the average distance between the cell adhesion factors along the polyisocyanopeptide backbone is 10-50 nm.

9. A method for inducing vascularization of stem cells, comprising the steps of: a) mixing a cell culturing medium for vascularization with an oligo(alkylene glycol) substituted co-polyisocyanopeptide at a temperature between 0 and 18 C. to obtain a polymer solution; b) mixing the polymer solution with stem cells at a temperature between 0 and 18 C. to obtain a cell culture solution; c) allowing the cell culture solution to warm to a temperature between 30 and 38 C. to form a cell culture comprising a hydrogel and allow the stem cells to differentiate, wherein a concentration of the polyisocyanopeptide in the polymer solution is 1-5 mg/ml, wherein an average length of the polyisocyanopeptide is 50-750 nm as determined by AFM, wherein a cell density of the stem cells in the cell culture solution is 0.3*10.sup.6-1*10.sup.6 cells/ml, wherein the hydrogel has a critical stress .sub.c of 2-12 Pa, preferably 7-12 Pa, wherein the critical stress is a stress which marks an onset of strain stiffening, wherein the hydrogel has a storage modulus G measured at 37 C. of 50-1000 Pa, preferably between 70-350 Pa, more preferably between 72-300 Pa and wherein the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide and/or wherein the cell culturing medium comprises fibrin, wherein when the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide, the average distance between the cell adhesion factors along the polyisocyanopeptide backbone is 10-50 nm.

10. A method for inducing adipogenic differentiation of stem cells, comprising the steps of: a) mixing a cell culturing medium for adipogenic differentiation with an oligo(alkylene glycol) substituted co-polyisocyanopeptide at a temperature between 0 and 18 C. to obtain a polymer solution; b) mixing the polymer solution with stem cells at a temperature between 0 and 18 C. to obtain a cell culture solution; c) allowing the cell culture solution to warm to a temperature between 30 and 38 C. to form a cell culture comprising a hydrogel and allow the stem cells to differentiate, wherein a concentration of the polyisocyanopeptide in the polymer solution is 1-5 mg/ml, wherein an average length of the polyisocyanopeptide is 50-750 nm as determined by AFM, wherein a cell density of the stem cells in the cell culture solution is 0.3*10.sup.6-1*10.sup.6 cells/ml, wherein the hydrogel has a critical stress .sub.c of 2-30 Pa, preferably 7-23 Pa or 8-20 Pa, wherein the critical stress is a stress which marks an onset of strain stiffening, wherein the hydrogel has a storage modulus G measured at 37 C. of 50-1000 Pa, preferably between 70-350 Pa, more preferably between 72-300 Pa, and wherein the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide and/or wherein the cell culturing medium comprises fibrin, wherein when the polyisocyanopeptide has a cell adhesion factor covalently bound to the polyisocyanopeptide, the average distance between the cell adhesion factors along the polyisocyanopeptide backbone is 10-50 nm, wherein the storage modulus G measured at 37 C. is 200-400 Pa and the viscosity average molecular weight (Mv) of the polyisocyanopeptide is between 100 and 1000 kg/mol, preferably 500-1000 kg/mol.

11. The method according to claim 8, wherein the storage modulus G measured at 37 C. is 200-400 Pa and the viscosity average molecular weight (Mv) of the polyisocyanopeptide is between 100 and 1000 kg/mol, preferably 200-700 kg/mol or 300-600 kg/mol.

12. The method according to claim 9, wherein the storage modulus G measured at 37 C. is 70-300 Pa and the viscosity average molecular weight (Mv) of the polyisocyanopeptide is between 100 and 1000 kg/mol, preferably 200-700 kg/mol or 300-600 kg/mol.

13. The method according to claim 10, wherein the storage modulus G measured at 37 C. is 70-450 Pa and the viscosity average molecular weight (Mv) of the polyisocyanopeptide is between 100 and 1000 kg/mol, preferably 200-700 kg/mol or 300-600 kg/mol.

14. The method according to claim 7, wherein the concentration of the polyisocyanopeptide in the polymer solution is 1.5-3 mg/ml.

15. The method according to claim 7, wherein the cell adhesion factor is covalently bound to the polyisocyanopeptide and wherein the polyisocyanopeptide is prepared by copolymerizing i) a first comonomer of an oligo(alkylene glycol) functionalized isocyanopeptide grafted with a linking group and a second comonomer of a non-grafted oligo(alkylene glycol) functionalized isocyanopeptide, wherein the molar ratio between the first comonomer and the second comonomer is 1:500 and 1:30; and ii) adding a reactant of a spacer unit and a cell adhesion factor to the copolymer obtained by step a), wherein the spacer unit is represented by general formula A-L-B; wherein the linking group and group A are chosen to react and form a first coupling and the cell adhesion factor and group B are chosen to react and form a second coupling, wherein the first coupling and the second coupling are independently selected from the group consisting of alkyne-azide coupling, dibenzocyclooctyne-azide coupling, oxanorbornadiene-based-azide couplings, vinylsulphone-thiol coupling, maleimide-thiol coupling, methyl methacrylate-thiol coupling, ether coupling, thioether coupling, biotin-strepavidin coupling, amine-carboxylic acid resulting in amides linkages, alcohol-carboxylic acid coupling resulting in esters linkages and NHS-Ester (N-Hydroxysuccinimide ester)-amine coupling and wherein group L is a linear chain segment having 10-60 bonds between atoms selected from C, N, O and S in the main chain.

16. The method according to claim 7, wherein the stem cells are chosen from human adipose stem cells and human mesenchymal stem cells.

17. The method according to claim 7, wherein the oligo(alkylene glycol) functionalized co-polyisocyanopeptide comprises a cell adhesion factor which is chosen from the group consisting of a sequence of amino acids of RGD, GRGDS, rhrVEGF-164 and rhrbFGF.

18. The cell culture according to claim 1, wherein the stem cells are chosen from human adipose stem cells and human mesenchymal stem cells.

19. The cell culture according to claim 3, wherein the oligo(alkylene glycol) functionalized co-polyisocyanopeptide comprises a cell adhesion factor which is chosen from the group consisting of a sequence of amino acids of RGD, GRGDS, rhrVEGF-164 and rhrbFGF.

20. (canceled)

21. (canceled)

22. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0226] FIG. 1 shows the relationship between differential modulus K and the applied stress for various hydrogels.

[0227] FIG. 2a shows the reaction of a non-functionalized monomer 1 and an azide appended monomer 2.

[0228] FIG. 2b shows the formation of BCN-GRGDS conjugate.

[0229] FIG. 2c shows the formation of polymer-peptide conjugates.

[0230] FIG. 2d shows the relationship of the stiffness G (Pa) of the hydrogel according to the invention and the temperature ( C.). The onset of gelation temperature was observed to be 15 C.

[0231] FIG. 2e shows the storage modulus G.sub.0 (Pa) of hydrogels P1-P6 in relation to the mean polymer length. The storage modulus (G.sub.o) remains fairly constant (0.2-0.4 kPa) at 37 C. as a function of polymer length.

[0232] FIG. 2f shows the critical stress.sub.c (Pa) in relation to the mean polymer length. The critical stress varies linearly as a function of polymer length.

[0233] FIG. 3a-c show images of the cells.

[0234] FIG. 3d-i show results of various tests for polymers P1-P6.

[0235] FIG. 4a-b shows the influence of the critical stress of the hydrogel to the stem cell differentiation.

[0236] FIG. 5 shows the critical stresses (94 .sub.c) of P1-P6 -MEM gels.

[0237] FIG. 6 shows the relationship between the molecular weight of the polyisocyanopeptides used according to the invention and the critical stress of the hydrogel made using the polyisocyanopeptides.

[0238] FIGS. 7-9 show the expression of osteogenic (RUNX2, ALP, FOSB and DLX5), endothelial (EDF1, VWF, KDR/FLK-1, and CD31), adipogenic (PPAR, CEBPB, LPL and FABP4) specific genes for stem cells grown in osteogenic, adipogenic and endothelial media, respectively; P7=soft, P8=medium, P9=hard.

[0239] FIG. 10 shows the growth of stem cells in alfa MEM (reference experiment); P7=soft, P8=standard, P9=hard.

DETAILED DESCRIPTION OF THE INVENTION

[0240] Experiment 1

[0241] Polyisocyanopeptides (P1-P6) were synthesized by a nickel (II)-catalyzed co-polymerization of triethylene glycol functionalized isocyano-(D)-alanyl-(L)-alanine monomer 1 and the azide-appended monomer 2 (FIG. 2a), with the molar ratio of 1/2=100, resulting in polymers with one azide functionality every 14-18 nm of the polymer chain, as determined by reacting a strained rhodamine dye to the azides (Table 1 and Methods).

[0242] The catalyst to monomer molar ratio was varied from 1:1000 to 1:8000, to obtain polymers of increasing molecular weight (determined by viscosity measurements, Table 1) (P1-P6). These azide functionalized polymers were then subjected to strain-promoted click reaction with BCN-GRGDS (BCN: Bicyclo[6.1.0]non-4-yn-9-ylmethyl) to obtain cell adhesive GRGDS functionalized polymers P1-P6 (FIG. 2b-c and Methods) of increasing chain lengths as determined by AFM (Table 1). Solutions of these polymers in -MEM (Minimum Essential Medium) at a fixed concentration (2 mg/mL) formed transparent gels upon warming, above 15 C. (temperature sweep rheology, FIG. 2d).

[0243] The mechanical properties of the GRGDS functionalized polymer gels were investigated by rheological analysis. Temperature sweep experiments (heating up to 37 C.) followed by time sweep at 37 C. revealed that all the gels P1-P6 were soft and exhibited similar stiffnesses (0.2-0.4 kPa at 37 C.) (FIG. 2e). Recently, we reported that hydrogels of non-functionalized polyisocyanopeptide polymers show a biomimetic stress stiffening behavior (Kouwer, P. H. J. et al. Responsive biomimetic networks from polyisocyanopeptide hydrogels. Nature 493, 651-655 (2013). Using the same pre-stress protocol (Broedersz, C. P. et al. Measurement of nonlinear rheology of cross-linked biopolymer gels. Soft Matter 6, 4120 (2010)), the critical stresses (.sub.c) of P1-P6 -MEM gels were measured (FIG. 5). The critical stress (.sub.c) for non-linear rheology behavior of these gels was found to increase linearly as a function of the polymer chain length (FIG. 2f and Table 1), from 9 Pa in the P1 gel (average polymer length: 182 nm) to 19 Pa in the P6 gel (average polymer length: 434 nm). Although it appears that there is a 1.5-fold increase in the mean gel stiffness when the mean polymer length is increased from 180 nm to 240 nm (FIG. 2e), this difference is small in the context of cellular perception of bulk stiffness.sup.17. Regarding the critical stress values, the error range is smaller and there appears to be a linear relationship between this parameter and the mean polymer length (FIG. 2f) which is why we consider the increase to be significant.

[0244] Effect of Stress-Stiffening on hMSC Commitment and Differentiation.

[0245] To investigate the effect of stress-stiffening on stem cell fate, hMSCs were mixed with a cold polymer solution (10 C.) in -MEM, which was then warmed to 37 C. to form the 3D matrix with encapsulated hMSCs. The cells were homogeneously distributed throughout the gel as indicated by confocal microscopy. Investigation of hMSCs morphology after 36 h of culture for all of the gels (P1-P6) revealed that the cells remained spherical (FIG. 3a). These cells exhibited only limited cortical F-actin protrusions into the surrounding microenvironment (Phalloidin staining) and showed no significant modifications in their nuclear morphology as shown by a representative DAPI fluorescence image of the cell nucleus after 36 h of culture (FIG. 3b). Live/dead assay (calcein-AM and MTT) performed after 36 h of culture in growth media for all of the gels indicated excellent viability (>95%) of the encapsulated cells (FIG. 3c and d), as also confirmed by confocal microscopy. In addition, no significant cell proliferation could be detected for the various gels as determined by the PicoGreen assay. The lineage commitment of the gel encapsulated hMSCs after 96 h of culture in bipotential differentiation medium (1:1 v/v osteogenic and adipogenic media) was then investigated. Cells were first stained (immunofluorescence) for STRO-1, a mesenchymal stem cell specific marker. A significant decrease in the average STRO-1 expression was observed for the cells in all of the gels after 96 h of culture, indicating the onset of stem cell differentiation (FIG. 3e). The expression of osteogenic and adipogenic differentiation markers was then examined. For cells cultured in the gel with the lowest critical stress (.sub.c 9.4 Pa, constructed from the shortest polymer P1) predominant adipogenic commitment was observed (Oil-red O staining, FIG. 3i). With increasing the critical stress (by increasing the polymer length), osteogenesis was progressively favored over adipogenesis, as demonstrated by immunofluorescent staining of Osterix, an osteogenic specific marker (FIG. 3h) and as determined from the mean percentages of osteogenic and adipogenic commitments in the various polymers (P1-P6). hMSCs cultured in the gel with the highest critical stress (P6) exhibited preferential osteogenic commitment. The predominant osteogenesis for the cells in the longer polymers (P4-P6) was further confirmed by differentiation tests after 3 weeks of culture.

[0246] Finally, the hMSCs osteogenic commitment was verified by analyzing the expression of the osteogenic biomarker Core-binding factor 1 (Cbfa-1), also called RUNX2 and the expression of the adipogenic biomarker PPAR, by RT-PCR. We observed an increase in the RUNX2 gene expression with increasing the critical stress after 96 h of culture (FIG. 3f) in agreement with the immunofluorescence staining results. Increase in osteogenesis for the longer polymer gels has been further confirmed by the observed decrease in PPAR gene expression as a function of the increasing critical stress after 96 h of culture (FIG. 3g).

[0247] To investigate the role of hMSCs-adhesive ligand interactions in the observed stem cell fate, we performed the cell commitment studies for RGD modified polymers P1, P3, P4 and P6 in the presence of antibodies recognizing specific integrin subunits (1, 2, 3 and 5; 31 and 2) which block their interactions with the substrate bound RGD ligands. In the presence of these integrin blocking antibodies, osteogenic commitment was suppressed. However, adipogenic commitment was maintained for all the polymers. This result is in agreement with recent literature and highlights the importance of the interaction between integrin receptors and the RGD ligands for mediating the stress-stiffening induced commitment switch. Interestingly, the presence of blebbistatin (a small molecule inhibitor of actomyosin contractility showing high affinity and selectivity toward myosin II) inhibited the hMSCs commitment, with sternness maintenance observed for all the polymer gels, as revealed by the high levels of STRO-1 in the encapsulated cells. This suggests that the inhibition of actomyosin contraction interferes with the mechanisms of hMSCs commitment both towards adipogenesis and osteogenesis. This is most likely due to the fact that the cells could not apply any traction force for the microenvironmental mechanical (stress-stiffening) sensing. These results are consistent with previously published studies. Finally, in order to demonstrate the direct interaction between the hMCSs and the polymer-bound RGD in our system, the cell commitment studies for RGD modified polymers P1, P3, P4 and P6 were performed in the presence of soluble RGD ligands, which can block the interaction between the cells and the matrix by competing for the integrin binding sites. No significant osteogenic or adipogenic commitment could be detected indicating that integrin disengagement from the matrix bound RGD is interfering with the cell's ability to sense stress-stiffening. These data also imply that the cells in these gel culture systems need direct engagement with the bound RGD ligand, and not with the secreted ECM, for mediating the stress-stiffening induced commitment switch.

[0248] Although the macroscopic ligand density is kept constant in this study (one ligand every 14-18 nm of a polymer chain), the longer polymer chains (P4-P6) have almost 2-fold higher number of ligands per chain (20-26), as compared to the corresponding shorter chains (P1-P3: 13-18). This could indeed impact the extent of cell-mediated local ligand clustering. To study the effect of ligand-density on the observed hMSC commitment switch, the commitment study was performed as a function of ligand density (RGD every 7 nm, 28 nm and 70 nm) for gels of the shortest (P1) and the longest polymer (P6). Varying the ligand density for both of the polymers was found not to interfere with the cell differentiation outcome. These results suggest that stress-stiffening is the primary governing variable in our system, without excluding the possibility that cell-mediated ligand clustering is occurring. Our data demonstrate that hMSCs fate can be switched from adipogenesis to osteogenesis in a soft microenvironment (0.2-0.4 kPa), simply by increasing the critical stress for the onset of stress-stiffening.

[0249] Stress-Stiffening Mediated Stem Cell Differentiation Involves the Microtubule-Associated Protein DCAMKL1.

[0250] Several reports have implicated the cytoskeletal contractility and actin polymerization in the mechanotransduction pathway responsible for osteogenic differentiation on 2D substrates. In our study, a treatment with cytochalasin-D (inhibitor of actin polymerization) resulted in an overall decreased commitment of the cultured stem cells towards both osteogenesis and adipogenesis, suggesting a role of actin polymerization in the stress-stiffening mediated hMSCs differentiation in our system. Alternatively we also observed a decrease in hMSCs commitment after treatment with Taxol, a well-characterized microtubule-stabilizing agent, which is known to inhibit tubulin de-polymerization. Taxol treatment did not affect cell viability as indicated by a live/dead assay after 48 h and 96 h of culture. The effect of Taxol on the cell commitment outcome indicates that, in addition to actin, the microtubule dynamics could also be involved in the mechanotransduction pathways underlying hMSCs differentiation in our system.

[0251] A recent report has indicated that the microtubule-associated protein DCAMKL1 represses RUNX2, an early osteogenesis marker, and thus regulates osteogenic differentiation in vitro and in an in vivo rat model. DCAMKL1 is also known to enhance microtubule polymerization. Furthermore, it has also been reported that microtubule de-polymerization can alter the myosin mechanochemical activity through myosin regulatory side chain phosphorylation, thus resulting in increased actomyosin contraction. We therefore investigated the role of DCAMKL1 in the stress-stiffening mediated control of hMSCs differentiation in our 3D culture system as a function of the gel critical stress. Interestingly, western blot analysis revealed a negligible DCAMKL1 expression for the polymer gel with the highest critical stress (P6) and a significant increase in the expression of this protein with decreasing the critical stress for stress-stiffening (FIG. 4a). Concomitantly, RUNX2 protein expression was not observed in the gels with lower critical stress (P1-P3) while the protein was clearly expressed in the higher critical stress polymers (P4-P6) in correlation with the observed osteogenic commitment in these gels. This is also in agreement with the observed overall increase in the RUNX2 mRNA expression between the shorter (P1-P3) and longer (P4-P6) polymers (FIG. 3f), although to a lesser extent, but still significant. These observations correlate well with preferential osteogenesis in gels of higher critical stress and lack of osteogenic commitment as the critical stress for stress-stiffening is lowered. A plot of the relative intensities (protein expression) of RUNX2 versus DCAMKL1 for all the conditions (P1 to P6) showed a switch-like relationship between these two proteins with the existence of a threshold value for the expression of DCAMKL1, which antagonizes RUNX2 in adipogenic lineage commitment (FIG. 4a). This observation has functional relevance for our mechanistic interpretations as it correlates with the observed stress-stiffening mediated commitment switch.

[0252] In order to further confirm the functional relationship between the two proteins in our stress-stiffening gel systems, DCAMKL1 gene silencing (through shRNA) and overexpression (via transient transfection) were performed for the hMSCs cultured in the P1 and P6 polymer gels. The DCAMKL1 silencing resulted in the increased expression of RUNX2 for the P1 polymer gel as well as for the P6 polymer gel but to a lesser extent. In contrast, DCAMKL1 overexpression did not significantly alter the expression of RUNX2 in the P1 polymer gel while a significant decrease was observed for P6. These data confirm the functional relationship between the two proteins in our gel system with DCAMKL1 being upstream of RUNX2 with a switch-like relationship, along with the existence of a threshold value for the expression of DCAMKL1, which inhibits the expression of RUNX2. In addition these data are in agreement with the previous in vivo and in vitro study.

[0253] Altogether these results are the first report of a microtubule-associated protein DCAMKL1 being involved in a new stress-stiffening mediated mechanotransduction pathway involving microtubule dynamics for the control of hMSCs differentiation (FIG. 4b). These data indicate that, stem cell fate is regulated by ECM stress stiffening via a different molecular mechanism than the one described for classical 2D substrate rigidity sensing.

[0254] Methods

[0255] AzideFunctionalized Polymer Synthesis (General Procedure). A solution of catalyst Ni(ClO.sub.4).sub.2.6H.sub.2O (1 mM) in toluene/ethanol (9:1) was added to a solution of non-functionalized monomer 1 and azide appended monomer 2 in freshly distilled toluene (50 mg/mL total concentration; molar ratio 1/2=100) in required amount and the reaction mixture was stirred at room temperature (20 C.) for 72 h. The resultant polymer was precipitated 3 times from dicholoromethane in di-isopropyl ether and dried overnight in air. The polymer was characterized by rheology, viscometry and AFM analysis. [0256] Synthesis of P1: The catalyst to monomer (1+2) molar ratio used: 1/1000 [0257] Synthesis of P2: The catalyst to monomer (1+2) molar ratio used: 1/2500 [0258] Synthesis of P3: The catalyst to monomer (1+2) molar ratio used: 1/3000 [0259] Synthesis of P4: The catalyst to monomer (1+2) molar ratio used: 1/4000 [0260] Synthesis of P5: The catalyst to monomer (1+2) molar ratio used: 1/6000 [0261] Synthesis of P6: The catalyst to monomer (1+2) molar ratio used: 1/8000

[0262] Conjugation of Azide-Functionalized Polymers with GRGDS Peptide: The GRGDS peptide was dissolved in borate buffer (pH 8.4) at a concentration of 2 mg/mL. A solution of BCN-NHS in DMSO was added to the peptide solution in borate buffer in 1:1 molar ratio and stirred on roller-mixer for 3 h at room temperature (20 C.). The formation of BCN-GRGDS conjugate was confirmed by mass spectrometry. MS calc.: 910.4, obtained: 911.4

[0263] The azide functionalized polymer (P1-P6) was dissolved in acetonitrile at a concentration of 3 mg/mL. To this solution, the appropriate volume of BCN-GRGDS solution in borate buffer (based on the molar equivalent of azide functions of the polymer) was added. The mixture was allowed to stir on roller-mixer for 72 h at room temperature (20 C.). The resultant polymer-peptide conjugates (P1-P6) were precipitated by adding the reaction mixture drop wise to di-isopropyl ether.

[0264] Determination of the Amount of Azides on the Azide Functionalized Polymer:

[0265] A dichloromethane solution of BCN conjugated lissamine dye was added to a dichloromethane solution of the polymer (1 mg/mL) in 1:1.2 molar ratio w.r.t. the calculated amount of azides in an azide polymer. The reaction mixture was rotated at 15 rpm in dark for 12 h at room temperature (20 C.). The polymer-dye conjugate was precipitated 4 times from dichloromethane in di-isopropylether, dried in air overnight, re-dissolved in dichloromethane, after which the absorption spectra were recorded. The extinction coefficient of 138,428 Lmol.sup.1cm.sup.1 was used at a wavelength of 559 nm to determine the amount of dye attached to the polymer, and thus to calculate the amount of azide present on the polymer (Table 1).

[0266] Rheology Analysis: The polymers were dissolved at a concentration of 2 mg/mL in -MEM (without serum) by gentle rotation (7-8 rpm) at 4 C. on a 90 rotor for 36 h. For determining the bulk stiffness of the gel, a variable temperature rheology was performed (plate-plate geometry; 250 m geometry gap), by heating the solution from 5 C. to 37 C. at a heating rate of 2 C./min at a constant strain of 2% and constant frequency of 1 Hz. This experiment was immediately followed by a time sweep experiment (5 min.) at 37 C. at a constant frequency of 1 Hz and the G observed at the end of the experiment was taken as the equilibrium bulk stiffness of the gel at this temperature. For non-linear rheology, the previously described pre-stress protocol was employed immediately after the aforementioned time sweep experiment.

[0267] The critical stress .sub.c was determined by the rheology analysis. For further details of determining the critical stress .sub.c, Jaspers, M. et al. Ultra-responsive soft matter from strain-stiffening hydrogels. Nat. Commun. 5, 5808 (2014), FIG. 2 and the section titled Mechanical analysis (p2-3) and the section titled Rheology (p7), incorporated by reference, are referred.

[0268] The critical stress .sub.c was determined by fitting (if possible) or by visual inspection of the obtained differential modulus (K) as a function of stress. Fitting was performed by fitting the non-linear regime to a single exponent (K=a.sup.m) to calculate .sub.c as the intercept between the fitted line and the region where K equals G.sub.0. When not enough data points could be recorded, the onset of deviation of linearity is taken as the .sub.c.

[0269] Atomic Force Microscopy: To visualize individual polymer chains and determine the average length of the polymers, solutions (1 g/mL inCHCl.sub.3) were spin coated (300 rpm for 20 seconds) on freshly cleaved mica substrates and imaged by using AFM tapping mode. Polymer lengths were determined by using the ImageJ software. The lengths of at least 150 polymer chains were counted to obtain the distribution and the mean of the polymer chain length for any particular sample.

[0270] Cell Culture. Human Mesenchymal Stem Cells (hMSCs) were obtained from Lonza, Inc. (Switzerland). Cells were then cultured in -MEM medium (lnvitrogen) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and incubated in a humidified atmosphere containing 5% (v/v) CO.sub.2 at 37 C. For the encapsulation of cells in the gels, first, the cell pellets were obtained by centrifugation. Then 500 L of the cold polymer solution (10 C.) was added directly to the pellet, followed by a gentle pipetting up and down 3-4 times to ensure a homogeneous mixture that was directly put onto a cover slip in a 6-well plate (also kept cold). Thereafter, the solution was sandwiched between two cover slips and the well plate was transferred to a 37 C. incubator. The volume of the suspension was chosen (500 L) in order to obtain hydrogel thickness in the range of 3 mm. The polymer solution forms a gel immediately after incubation at 37 C. as revealed by kinetic rheology experiments. Afterwards, the gel becomes stiffer with time and attains the final stiffness in 2-3 minutes. This favors the supporting of cells in 3D rather than the cells settling at the bottom. After gel formation, the two cover slips were removed and -MEM medium (without serum) was added. All cell culture experiments were carried out without any serum in the medium for the first 6 h of culture. Then, -MEM medium with 10% serum was added. All cells were used at low passage numbers (passage 4), were subconfluently cultured and were seeded at 10.sup.6 cells/mL for the purpose of the experiments and in order to avoid cell-cell contacts. The lineage commitment and differentiation of the gel encapsulated hMSCs after 96 h and 3 weeks of culture, respectively, were investigated with bipotential differentiation medium (1:1 v/v osteogenic and adipogenic media, Lonza). For all the experiments, a non-functionalized soft polymer gel (cell culture in growth medium) served as control. The live/dead viability assay at 3 weeks in these control gels indicated excellent cell viability. The pharmacological agents used were 50 M Blebbistatin (EMD Biosciences-Calbiochem), 1 M cytochalasin D (Sigma) and 50 nM Taxol (Abcam). The hMSCs were exposed to each pharmacological agent for 1 h, 24 h and 72 h, respectively, after seeding on a modified polymer. For antibody inhibition studies, cells were preincubated with 5 ng/mL anti-1, 2, 3 and 5-1, 2 (all from Santa Cruz Biotechnology). For competition experiments with soluble RGD peptides, the cells were incubated in 1 mL of cell culture media containing 200 g of RGDS peptides during 20 min on plastic and then transferred to the polymer gels. To evaluate proliferation, total double-stranded DNA content was determined by using the PicoGreen assay as previously reported.

[0271] Confocal Microscopy. In order to assess the homogeneous distribution of cells in our hydrogels, very thin slices of the gel were cut transversely at various depths, including the two interfaces. The fluorescently labeled cells encapsulated in the gel slices were imaged by confocal microscopy with a Leica SP5 confocal microscope, 10 objective, 0,3 NA. 400 m thick z-stacks were then acquired every 2,39 m and the 3D images were reconstructed by using the Imaris 7.0 software.

[0272] MTT Assay. As described in literature, briefly, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the data are presented as a percentage of control viability.

[0273] Live/Dead Staining. Cell viability was determined with the live/dead viability/cytotoxicity kit (Molecular Probes), according to the manufacturer's protocol.

[0274] Real-Time PCR Analysis of Gene Expression. RT-PCR was performed as previously described. Briefly, total RNA was extracted by using the RNeasy total RNA kit from Qiagen in accordance with the manufacturer's instructions. Purified total RNA was used to make cDNA by reverse transcription reaction (Gibco BRL) by using random primers (Invitrogen). Real-time PCR was performed by using SYBR green reagents (Bio-Rad). The data were analyzed by using the iCycler IQTM software. The cDNA samples (1 L in a total volume of 20 L) were analyzed for the gene of interest and for the house-keeping gene GAPDH. The comparison test of the cycle-threshold point was used to quantify the gene expression level in each sample. The primers used for the amplification are listed in Supplementary Table 1.

[0275] Western Blotting. After 96 h, the polymer gels were exposed to a cold environment (around 10 C.). The cell pellet was obtained by centrifugation. The cells were permeabilized (10% SDS, 25 mM NaCl, 10 nM pepstatin and 10 nM leupeptin in distilled water and loading buffer), boiled for 10 min and resolved by reducing PAGE (Invitrogen). Proteins were transferred onto nitrocellulose, blocked, and labeled with HRP-conjugated antibodies (Invitrogen). The microtubule associated protein DCAMKL1 was blotted by using the monoclonal anti-DCAMKL1 antibody (Santa Cruz Biotechnology). The transcriptional factor RUNX2 was blotted by using the monoclonal anti-Runx2 antibody (Abcam). The western blots in these experiments were run in triplicate, along with an additional blot for tubulin and Coomassie Blue staining to ensure consistent protein load between samples. In order to construct the plot of the relative intensities of RUNX2 versus DCAMKL1 (FIG. 4a) and to illustrate the switch-like relationship between the two proteins, the zero of the RUNX2 relative intensity was set at the corresponding level of expression of RUNX2 in hMSCs cultured on plastic, which was set to 1.

[0276] Immunostaining. After 96 h of culture, the gels were exposed to cold environment (10 C.), the cell pellet was collected from the fluid by centrifugation, transferred onto the well plate and allowed to adhere to the well plate surface by culturing in A-MEM with serum for 16 h. The cells were then fixed for 20 min in 4% paraformaldehyde/PBS at 37 C. After fixation, the cells were permeabilized in a PBS solution of 1% TritonX100 for 15 min. The cells were then incubated with primary antibody (mouse anti-vinculin for adhesion, mouse anti-STRO-1 for differentiation) for 1 h at 37 C. After washing, cells were stained with Alexa Fluor 647 rabbit anti-mouse IgG secondary antibody for 30 min. at 37 C. Cell cytoskeletal filamentous actin (F-actin) was visualized by treating the cells with 5 U/mL Alexa Fluor 488 Phalloidin (Sigma, France) for 1 h at 37 C. Vinculin was visualized by treating the cells with 1% (v/v) monoclonal anti-vinculin (clone hVIN-1 antibody produced in mouse) for 1 h at 37 C. The cells were then stained with Alexa fluor 568 (F(ab)2 fragment of rabbit anti-mouse IgG(H+L)) during 30 min at room temperature. After 96 h, Osterix was visualized by treating the cells with 1% (v/v) rabbit monoclonal anti-Osterix (antibody produced in rabbit) for 1 h at 37 C. The cells were then stained with Alexa fluor 568 (F(ab)2 fragment of mouse anti-rabbit IgG(H+L)) during 30 min at room temperature. Tubulin (stained by Anti-Tubulin 3 (Sigma, France) was visualized by treating the cells with 1% (v/v) monoclonal anti-Tubulin 3 (Abcam, Cambridge), for 1 h at 37 C. and then with Alexa Fluor 588 (F(ab)2 fragment of goat anti-rabbit IgG(H+L)) for 30 min at room temperature. There was no detection of the muscle transcription factor MyoD1 (stained with anti-MyoD1 (Santa Cruz Biotechnology, USA)). To stain lipid fat droplets, the cells were fixed in 4% paraformaldehyde, rinsed in PBS and 60% isopropanol, stained with 3 mg ml.sup.1 Oil Red O (Sigma, France) in 60% isopropanol and rinsed in PBS at 37 C.

[0277] For quantification of STRO-1, Osterix, Tubulin 3, MyoD1 and lipid fat droplets, positive contacts number and areas, we used the freeware image analysis ImageJ software. First the raw image was converted to an 8-bit file, and the unsharp mask feature (settings 1:0.2) was used to remove the image background (rolling ball radius 10). After smoothing, the resulting image, which appears similar to the original photomicrograph but with minimal background, was then converted to a binary image by setting a threshold. The threshold values were determined by selecting a setting, which gave the most accurate binary image for a subset of randomly selected photomicrographs with varying glass substrates. The total contact area and mean contact area per cell were calculated by analyse particules in Image J. A minimum of 20 to 30 cells per condition were analyzed.

[0278] Statistical Analysis. In terms of fluorescence intensity, sub-cell contact area and real-time PCR assay, the data were expressed as the meanstandard error, and were analyzed by using the paired Student's t-test method. Significant differences were designated for P values of at least <0.01.

[0279] Overexpression of DCAMKL1. The overexpression of DCAMKL1 was performed as previously described by Lin PT et a1.sup.57. Briefly, Human DCAMKL1 was cloned by RT-PCR using primers directed toward the human sequence and was subsequently sequenced. Full-length human DCAMKL1 was subsequently cloned into the Kpnl site of pcDNA3.1 C() (Invitrogen, Carlsbad, Calif.) and overexpressed by transient transfection with Super-fectamine (Qiagen, Chatsworth, Calif.) according to the manufacturer's recommendations. The efficiency of the DCAMKL1 overexpression was assessed by western blot for hMSCs cultured on plastic. A 180-200% increase in protein level was observed after 72 h.

[0280] DCAMKL1 shRNA Silencing. DCAMKL1 silencing has been performed by transfecting hMSCs with a pool of 3 target-specific lentiviral vector plasmids each encoding 19-25 nt (plus hairpin) shRNAs designed to knock down gene expression (Santa Cruz Biotechnology). A mock plasmid was transfected as a control. Transient transfection was performed by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The efficiency of the DCAMKL1 silencing was assessed by western blot for hMSCs cultured on plastic. The DCAMKL1 silencing decreased DCAMKL1 mRNA level by 50-60% (not shown) and DCAMKL1 protein level by 60-70% after 24 h.

TABLE-US-00001 TABLE 1 Properties of oligo(alkylene glycol) functionalized co-polyisocyanopeptide P1-P6 Viscosity Mean Mean Critical derived Average (GRGDS Stress molecular spacing of functionalized) (.sub.c, Pa) in - weight N3 on the polymer MEM gels at Catalyst/ (N3-polymer; polymer length from 2 mg/mL Polymer monomer kg/mol) chain (nm) AFM (nm) concentration P1 1/1000 307 14 182 9.4 P2 1/2500 426 14 226 9.9 P3 1/3000 491 18 250 12.8 P4 1/4000 571 15.6 309 14.6 P5 1/6000 591 14 367 16.6 P6 1/8000 685 17 434 19.3

[0281] It was observed that the use of polymers P1-P3 led to adipogenic differentiation whereas the use of polymers P4-P6 led to osteogenic differentiation.

[0282] Experiment 2

[0283] Oligo(alkyleneglycol)-substituted polyisocyanopeptides were prepared by using various ratios catalyst/monomer as shown in table 1. GRGDS was used as the cell adhesion factor. The decrease in the catalyst/monomer ratio resulted in an increase in viscosity average molecular weight (Mv) and the mean polymer length, while the distribution of the cell adhesion factor over the polymer chain remained at a constant level of 1 cell adhesion factor per 14-18 nm of polymer backbone. The relationship between the molecular weight and the mean polymer length can also be derived from table 1.

[0284] FIG. 6 shows the relationship between the molecular weight of the polyisocyanopeptides used according to the invention and the critical stress of the hydrogel made using the polyisocyanopeptides.

[0285] Experiment 3

[0286] Polymer Preparation

[0287] Polyisocyanopeptides (P7-P9) were synthesized as described above.

[0288] The catalyst to monomer molar ratio was 1:1000, 1:5000 and 1:7000 respectively, to obtain polymers of increasing molecular weight (determined by viscosity measurements, Table 2) (P7-P9). These azide functionalized polymers were then subjected to strain-promoted click reaction with BCN-RGD10 (BCN: Bicyclo[6.1.0]non-4-yn-9-ylmethyl) to obtain cell adhesive RGD10 functionalized polymers (PIC-RGD10) P7-P9. The strain-promoted click reaction is performed in the same way as described for functionalization with BCN-GRGDS under Methods above.

TABLE-US-00002 TABLE 3 Properties of oligo(alkylene glycol) functionalized co-polyisocyanopeptide P7-P9 G @37 C., Code Polymer .sub.c, Pa LOST, C. Pa** Mv, kDa P7 RGD10 1k 7* 18 78 375 P8 RGD10 5k 18* 15 230 545 P9 RGD10 7k 23.6 14 214 614 *Plate slipping/Gel braking resulting in not enough data points for fitting to obtain .sub.c decimals. Values obtained by visual inspection of the data. **The G values are measured in incomplete -MEM.

[0289] The average viscosity molecular weight, M, of the polymers was calculated using the empirical Mark-Houwink equation, []=KM.sub.v.sup.a, where [] is the intrinsic viscosity of the polymer solution (in acetonitril) as determined from viscometry measurements, using a Ostwald tube, and Mark-Houwink parameters K and a depend on polymer and solvent characteristics. We used values that were previously determined for (other) rigid polyisocyanides: K=1.4109 and a=1.75 (Van Beijnen, A., Nolte, R., Drenth, W., Hezemans, A. & Van de Coolwijk, P. Helical configuration of poly(iminomethylenes). Screw sense of polymers derived from optically active alkyl isocyanides. Macromolecules 13, 1386-1391 (1980).)

[0290] Effect of Stress-Stiffening on hASC Differentiation

[0291] Human adipose derived stem cells (hASCs, passage 3) were cultured in MEM (Sigma, Germany) supplemented with 10% fetal calf serum (FCS) and 1% Penicilin/Streptomycin (P/S), until reaching 70% confluence. The cells were trypsinized and prepared in a suspension of 10.sup.6 cells in complete MEM. Equal volumes of cells suspension and cold PIC-RGD10 solution, previously prepared at 4 mg/ml in complete MEM, were slowly mixed until cells were evenly distributed within the gel, thus rendering a 2 mg/mL gel suspension containing 0.5106 cells/ml. Three different PIC-RGD10 batches with different stiffness (soft, intermediate, hard) were used for encapsulation of hASCs. 150 uL of the gel-hASCs suspension were carefully loaded into 48-well plates wells allowed to solidify at 37 C. After 10 minutes, 200 uL of warmed MEM were gently added to each well and cultured overnight at 37 C. and 5% CO.sub.2. The next day, used media was replenished with different media, depending on the experiment.

[0292] osteogenic differentiation medium (OST) consisting of complete MEM, 50 mM -glycerophosphate anhydrous, 50 g/ml ascorbic acid and 10.sup.8 M dexamethasone (results FIG. 7)

[0293] commercially available adipogenic differentiation medium (ADIPO, Stemcell technologies, Cat Nr. 05412) Results FIG. 8

[0294] endothelial differentiation medium (ENDO) consisting of DMEM high glucose supplemented with 50 ng/mL recombinant vascular endothelial growth factor (rhVEGF) and 10 ng/mL recombinant basic fibroblast growth factor (rhbFGF), 2% FCS and 1% P/S (results FIG. 9)

[0295] complete MEM (control medium) (results FIG. 10)

[0296] Cells were allowed to grow in the gels for 21 days with replenishment of media every 3 days. At days 3, 7, 14 and 21, samples were retrieved and stored in 800 L TRIzol reagent (Life Technologies) for mRNA extraction and conversion to cDNA. Real time RT-PCR reactions were carried out for osteogenic (RUNX2, ALP, FOSB and DLX5), endothelial (EDF1, VWF, KDR/FLK-1, and CD31), adipogenic (PPAR, CEBPB, LPL and FABP4) and sternness (STRO1, ENG, NT5E and THY-1)specific genes.

[0297] The media composition triggers the differentiation, while material properties of the RGD10-functionalized polyisocyanopeptides (P7-P9) (such as stiffness, RGD10 content, etc) support and enhance certain differentiation pathways.

[0298] In FIG. 7a-d can be seen that osteogenic differentiation of the stem cells is primarily supported by hydrogels of polymer P9 with the highest viscosity and the highest critical stress.

[0299] According to FIG. 8a-d, the adipogenic differentiation of the stem cells is supported in the environment of hydrogels of polymers P8 and P9, with a medium to high viscosity and a medium to high critical stress.

[0300] FIG. 9a-d shows that the endothelial differentiation is supported primarily in the environment of hydrogels of polymer P8 with a medium viscosity and a medium value of the critical stress.

[0301] The cell morphology of the hASCs in a non-differentiating -MEM cell growth medium combined with the three different PIC-RGD10 batches with different stiffness (soft, intermediate, hard) was studied for 15 days. FIG. 10 shows photographs of the hASCs in the hydrogels over time.

[0302] In FIG. 10 it can be observed that the cells grow fast in the soft hydrogel and slower in the intermediate (standard) hydrogel. Growth of the hASCs is shown by the stretched morphology of the cells. In the hard hydrogel the cells did not grow and remained round.

[0303] In experiment 3, the cells are grown in a single differentiation medium, while in experiment 1 the cells are grown in a bipolar medium, which gives the cells the opportunity to grow and differentiate in two directions: either adipogenic or osteogenic.