Gelation Characteristics and Osteogenic Differentiation of Stromal Cells in Inert Hydrolytically Degradable Micellar Polyethylene Glycol Hydrogels

The use of poly­(ethylene glycol) (PEG) hydrogels in tissue engineering is limited by their persistence in the site of regeneration. In an attempt to produce inert hydrolytically degradable PEG-based hydrogels, star (SPELA) poly­(ethylene glycol-<i>co</i>-lactide) acrylate macromonomers with short lactide segments (<15 lactides per macromonomer) were synthesized. The SPELA hydrogel was characterized with respect to gelation time, modulus, water content, sol fraction, degradation, and osteogenic differentiation of encapsulated marrow stromal cells (MSCs). The properties of SPELA hydrogel were compared with those of the linear poly­(ethylene glycol-<i>co</i>-lactide) acrylate (LPELA). The SPELA hydrogel had higher modulus, lower water content, and lower sol fraction than the LPELA. The shear modulus of SPELA hydrogel was 2.2 times higher than LPELA, whereas the sol fraction of SPELA hydrogel was 5 times lower than LPELA. The degradation of SPELA hydrogel depended strongly on the number of lactide monomers per macromonomer (nL) and showed a biphasic behavior. For example, as nL increased from 0 to 3.4, 6.4, 11.6, and 14.8, mass loss increased from 7 to 37, 80, 100% and then deceased to 87%, respectively, after 6 weeks of incubation. The addition of 3.4 lactides per macromonomer (<10 wt % dry macromonomer or <2 wt % swollen hydrogel) increased mass loss to 50% after 6 weeks. Molecular dynamic simulations demonstrated that the biphasic degradation behavior was related to aggregation and micelle formation of lactide monomers in the macromonomer in aqueous solution. MSCs encapsulated in SPELA hydrogel expressed osteogenic markers Dlx5, Runx2, osteopontin, and osteocalcin and formed a mineralized matrix. The expression of osteogenic markers and extent of mineralization was significantly higher when MSCs were encapsulated in SPELA hydrogel with the addition of bone morphogenetic protein-2 (BMP2). Results demonstrate that hydrolytically degradable PEG-based hydrogels are potentially useful as a delivery matrix for stem cells in regenerative medicine

CSC fraction of MDA231 cells encapsulated in the patterned 5 kPa gel.

<p>MDA231 cells cultured on adherent tissue culture plate (TCP, A), non-adherent tissue culture plate (suspension, B), by encapsulation in Matrigel (C), encapsulation in un-patterned 5 kPa PEGDA gel (D), and encapsulation in 50 μm patterned 5 kPa PEGDA gel; (F) CSC fraction of the MDA231 cells in (A-E) as the sub-population of cells in the fourth quadrant (CD44⁺/CD24^-).</p

Dependence of tumorsphere growth on culture medium in 2D versus 3D for MDA231 cells.

<p>Number density (A) and mRNA expression of CD44 (B), ABCG2 (C), and EGFR (D) markers for MDA231 cells as a function of incubation time. Groups included cells on 2D adherent plates and cultured in RPMI-1640 medium (2D-RPMI), cells on 2D plates and cultured in CSC medium (2D-CSC), and cells encapsulated in the 5 kPa PEGDA gel and cultured in CSC medium (3D-CSC). An Asterisk in (A) indicates a statistically lower (p<0.05) cell number in the test group compared to 2D groups at the same time point. An Asterisk in (B-D) indicates a statistically higher (p<0.05) mRNA expression in the test group compared to 2D groups at the same time point. The p-values for the asterisks in (A-D) are listed in Tables E-H in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132377#pone.0132377.s001" target="_blank">S1 File</a>. Error bars correspond to means±1 SD for n = 3.</p

Dependence of the optimum gel modulus for CSC growth on tissue origin of cancer cells.

<p>(A) DAPI (blue) and phalloidin (red) stained images of MCF10A, MCF7, MDA231, HCT116, U2OS, and AGS cancer cells encapsulated in the un-patterned gels after 2, 6, and 9 days of incubation (scale bars in A are 50 μm). Cell number (B), tumorsphere number (C), tumorsphere diameter (D), and tumorsphere size distribution (E) as a function of cancer cell type after 9 days of encapsulation for gel moduli of 2 (pink), 5 (blue), 25 (brown), 50 (purple), and 70 (green) kPa. The gel modulus in (A and E) was the optimum PEGDA modulus of 5 kPa for MCF7 and MDA231 cells; 25 kPa for HCT116 and AGS; 50 kPa for U2OS. (F) mRNA expression of CSC markers for MCF7 (CD44 and ABCG2), MDA231 (CD44 and EGFR), HCT116 (CD44 and TGF-β), U2OS (CD44 and CD133), and AGS (CD44 and OCT4) after 6 and 9 days of encapsulation for gel moduli of 2 (pink), 5 (blue), 25 (brown), 50 (purple), and 70 (green) kPa. An asterisk in (B-D) indicates a statistically higher (p<0.05) cell number, sphere number and size for the test modulus compared to all other moduli for a given cell type. An asterisk in (F) indicates a statistically higher (p<0.05) marker expression level for the test modulus compared to all other moduli for a given cell type and at a given time. The p-values for the asterisks in (B-D,F) are listed in Tables I-U in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132377#pone.0132377.s001" target="_blank">S1 File</a>. Error bars correspond to mean±1 SD for n = 3.</p

Optimum 3D Matrix Stiffness for Maintenance of Cancer Stem Cells Is Dependent on Tissue Origin of Cancer Cells

<div><p>Introduction</p><p>The growth and expression of cancer stem cells (CSCs) depend on many factors in the tumor microenvironment. The objective of this work was to investigate the effect of cancer cells’ tissue origin on the optimum matrix stiffness for CSC growth and marker expression in a model polyethylene glycol diacrylate (PEGDA) hydrogel without the interference of other factors in the microenvironment.</p><p>Methods</p><p>Human MCF7 and MDA-MB-231 breast carcinoma, HCT116 colorectal and AGS gastric carcinoma, and U2OS osteosarcoma cells were used. The cells were encapsulated in PEGDA gels with compressive moduli in the 2-70 kPa range and optimized cell seeding density of 0.6x10⁶ cells/mL. Micropatterning was used to optimize the growth of encapsulated cells with respect to average tumorsphere size. The CSC sub-population of the encapsulated cells was characterized by cell number, tumorsphere size and number density, and mRNA expression of CSC markers.</p><p>Results</p><p>The optimum matrix stiffness for growth and marker expression of CSC sub-population of cancer cells was 5 kPa for breast MCF7 and MDA231, 25 kPa for colorectal HCT116 and gastric AGS, and 50 kPa for bone U2OS cells. Conjugation of a CD44 binding peptide to the gel stopped tumorsphere formation by cancer cells from different tissue origin. The expression of YAP/TAZ transcription factors by the encapsulated cancer cells was highest at the optimum stiffness indicating a link between the Hippo transducers and CSC growth. The optimum average tumorsphere size for CSC growth and marker expression was 50 μm.</p><p>Conclusion</p><p>The marker expression results suggest that the CSC sub-population of cancer cells resides within a niche with optimum stiffness which depends on the cancer cells’ tissue origin.</p></div

Dependence of tumorsphere growth on EMT marker expression for MDA231 cells.

<p>mRNA expression of EMT markers E-cadherin (A), N-cadherin (B), TGF-β (C), Snail (D), Slug (E), Twist (F), Vimentin (G), and ZEB2 (H) for MDA231 cells encapsulated in the un-patterned 5 kPa gel as a function of incubation time. Protein expression of pYAP (I), total YAP (J), YAP/TAZ (K) for the MDA231 cells encapsualted in the un-patterned gel as a function of modulus after 6 days of incubation. An asterisk in (A) indicates a statistically lower marker expression for that time point compared to all other time points. An asterisk in (B,D-H) indicates a statistically higher marker expression for that time point compared to all other time points. An asterisk in (I) indicates a statistically lower protein expression for that modulus compared to the other two moduli. An asterisk in (k) indicates a statistically higher protein expression for that modulus compared to the other two moduli. The p-values for the asterisks in (A,B,D-I,K) are listed in Tables G-O in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132377#pone.0132377.s002" target="_blank">S2 File</a>. Error bars correspond to mean±1 SD for n = 3.</p

Dependence of tumorsphere growth on conjugation of CD44 binding peptide to the gel.

<p>(A) DAPI (blue) and phalloidin (red) stained images of MDA231 (A+B), HCT116 (C+D), and U2OS (E+F) cells encapsulated in the un-patterned gel with optimum modulus (5 kPa for MDA231, 25 kPa for HCT116, and 50 kPa for U2OS) without CD44BP (A+C+E) and with CD44BP conjugation (B+D+F) after 9 days incubation (scale bar in A-F is 200 μm). The initial seeding density of all cell types in the gel was 0.6x10⁶ cells/mL. Cell number (G), tumorsphere size (H), and tumorsphere number (I) for MDA231, HCT116, and U2OS cells encapsulated in the gel without (blue) and with (green) conjugated CD44BP and with conjuagted mutant-CD44BP (mCD44BP, orange) after 9 days incubation. mRNA expression of CSC markers for MDA231 (J, CD44 and EGFR), HCT116 (K, CD44 and TGF-β), and U2OS (L, CD44 and CD133) encapsulated in the gel without conjugation, with mCD44BP, and with CD44BP conjugation. An Asterisk in (G-L) indicates a statistically lower cell number, sphere number and size, and marker expression for the test group compared to those groups without CD44BP conjugation and with mutant CD44BP conjugation of the gel for a given cell type. The p-values for the asterisks in (G-L) are listed in Tables A-F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132377#pone.0132377.s002" target="_blank">S2 File</a>. Error bars correspond to mean±1 SD for n = 3.</p

Dependence of tumorsphere growth on niche size for MDA231 cells.

<p>(A) Procedure for cell encapsulation in the micropatterned gel. CellTracker stained images of MDA231 cells encapsulated in the 5 kPa gel with circular patterns with diameter of 50 (B), 75 (C), 100 (D), 150 (E), and 250 (F) μm after 2 days of incubation (scale bars in B-F are 200 μm). DAPI (blue) and phalloidin (red) stained images of 7 representative tumorspheres formed by the encapsulated cells in (B-F) from different sections of the patterned gels after 14 days of incubation with 50 (G), 75 (H), 100 (I), 150 (J), and 250 (K) μm patterns (scale bars in G-K are 50 μm). Note that the 7 representative tumorspheres in (G-K) are from multiple patterns in the gel sample, not a single pattern, to show size range and shape of tumorspheres for a given pattern size. Cell number (L), average tumorsphere size (M), CD44 expression (N), and ABCG2 expression (O) of the cells in (B-F) with incubation time for 50 (pink), 75 (blue), 100 (red), 150 (green), 250 (brown) μm patterns, and un-patterned gel (purple). Error bars in (L-O) correspond to mean±1 SD for n = 3.</p

Forward and reverse sequence for the PCR primers.

<p>Forward and reverse sequence for the PCR primers.</p

Effect of CD44BP conjugated to the gel on tumor formation <i>in vivo</i>.

<p>The gel without cell (negative control, light blue), 4T1 tumospheres in suspension (positive control, red), 4T1 cells encapsulated in the gel without CD44BP (green), and 4T1 cells encapsulated in the gel with CD44BP (light blue) were inoculated subcutaneously in syngeneic Balb/C mice. Tumor sizes were measured daily from post-inoculation day 11 (n = 6/group). Tumor growth was not observed in the negative control group (the gel without cell) and the group with 4T1 cells in the gel with CD44BP (the lines for these two groups are overlapped in the figure).</p