Supplemental Table 1.The Composition of Each Polymer Solution Used in the Synthesis Of
Supplemental Table 1.The composition of each polymer solution used in the synthesis of substrates.
Sample / Solute Concentration / Solvent CompositionPLA / 9% wt. / CF/DMF (w/w=4/1)
PPC / 10% wt. / CF/DMF (w/w=4/1)
PCL / 12% wt. / CF/DMF (v/v=3/2)
TPU / 11% wt. / DMF
Supplemental Table 2. Antibodies and stains used for immunocytochemistry.
Target / Vendor/Cat# / DilutionTRA-1-60 / Millipore MAB4360 / 1:100
CD44 / BD 560531 / 1:200
Nanog / R&D Systems AF1997 / 1:200
SSEA-4 / Millipore MAB4304 / 1:50
Oct3/4 / BD 560186 / 1:10
Sox2 / BD 560531 / 1:40
F-Actin / Sigma P1951 / 1:400
Nuclei / Life Technologies H1399 / 1:10,000
Supplemental Table 3.All properties characterized for our materials library.Mean values ± 95% confidence intervals are listed for all values except fiber alignment which is listed as mean values ± standard deviation.AllTCPS data refers to 24 well plate dimensions. A fiber alignment of zero degrees would indicate perfect alignment. The average fiber diameter is not applicable to TCPS. We were unable to fabricate aligned fibers with the proper requirements for water contact angle, surface area analysis and tensile testing. N/A, non-applicable.
TCPS / PLA-R / PLA-A / PCL-R / PCL-A / TPU-R / TPU-A / PPC-R / PPC-A / PCL-SK-R / PCL-SK
-A
Fiber Diameter (nm) / N/A / 960
±76 / 788
±93 / 617
±84 / 537
±59 / 594
±29 / 669
±59 / 829
±50 / 794
±66 / 980
±80 / 990
±63
% Porosity / N/A / 38±6 / 65±4 / 49±4 / 84±3 / 43±5 / 73±9 / 45±0.16 / 75±15 / 26.71±5 / 38±10
BET Surface Area (m2/g) / N/A / 4.168 / 4.168 / 2.201 / 2.201 / 2.129 / 2.129 / N/A / N/A / N/A / N/A
Surface area per substrate (cm2) / 1.9 / 1724 ±355 / 1690 ±5 / 808 ±107 / 684 ±5 / 26 ±7 / 6 ±2 / N/A / N/A / N/A / N/A
Elastic Modulus (MPa) / 533 ±47 / 40 ±1.5 / N/A / 10.2 ±1.5 / N/A / 7.4 ±0.1 / N/A / 432.3 ±15.4 / N/A / 10.5 ±1.7 / N/A
Elongation at break (%) / 5.1 ±2.0 / 71.7 ±9.3 / N/A / 148.6 ±3.8 / N/A / 150.9 ±11.2 / N/A / 1285.8 ±240.8 / 1197.9 ±142.6 / N/A / N/A
Protein Adsorption (µg/cm2) / 21.2 ±4.0 / 1.33 ±0.2 / 0.779 ±0.1 / 2.03 ±0.3 / 1.77 ±0.1 / 71.3 ±11.3 / 192.3 ±9.5 / 1285 ±240 / 1197 ±142 / N/A / N/A
Fibroblast Proliferation Index / 1.29 ±0.56 / 2.19 ±1.26 / 1.55 ±0.59 / 2.01 ±1.19 / 0.78 ±0.28 / 1.61 ±1.15 / 1.89 ±1.96 / 1.59 ±1.27 / 2.52 ±1.38 / 3.79 ±2.85 / 1.01 ±0.54
Fibroblasts Attachment Day 1
(total cells) / 28,907±3098 / 10,927 ±5,186 / 8,077 ±2,162 / 12,289±5,531 / 12,717±3,401 / 16,111±4,645 / 14,103±10,600 / 13,183 ±7,285 / 14,340 ±6,196 / 14,659 ±6282 / 7,929 ±3,486
Stem Cell Proliferation Index / 8.81 ±1.71 / 3.34 ±1.87 / 2.47 ±1.14 / 3.14 ±1.32 / 6.72 ±4.22 / 3.54 ±0.38 / 4.61 ±2.74 / 2.83 ±1.28 / 3.01 ±1.49 / 3.49 ±1.14 / 3.72 ±0.96
Stem Cell Attachment Day 1
(total cells) / 7,246 ±233 / 5,052 ±2,664 / 4,844 ±1,882 / 8,768 ±2,264 / 4,782 ±2,664 / 7,724 ±811 / 7,416 ±3,697 / 7,483 ±1,268 / 8,386 ±2,611 / 5,551 ±1704 / 5,758 ±1,396
Water Contact Angle (Degrees) / 65
±6.1 / 116 ±6.5 / N/A / 127
±1.1 / N/A / 111
±1.3 / N/A / 112
±0.2 / N/A / 128
±0.4 / N/A
Supplemental Figure 1 - Cordie
Supplemental Figure 1.Additional nanofibroussubstrates with attached fibroblasts.Electron microscopy images of polycaprolactone-shish kebab (PCL-SK),andpoly(propylene carbonate) (PPC)nanofibers. Below each image is an immunocytochemistry image of HEF1 fibroblasts grown on each substrate. A control image was taken of HEF1 fibroblasts grown on tissue culture polystyrene (TCPS). Cells were fixed after five days in culture. Nuclei and F-actin were stained by Hoechst dye and phalloidin, respectively. Arrows indicate direction of fiber alignment.
Supplemental Figure 2 - Cordie
Supplemental Figure 2.Distribution of nanofiber diameter and alignment.Histograms of fiber diameter and fiber alignment for each nanofibrous substrate constructed. Measurements were gathered byImageJ analysis of SEM images. A fiber alignment of zero degrees would indicate perfect alignment.
Supplemental Figure 3 - Cordie
Supplemental Figure 3.Protein adsorption to nanofibrous substrates. A) Total protein adsorption to each substrate. B) Protein adsorption normalized by substrate surface area. Protein was allowed to adsorb to substrates overnight in standard cell culture conditions and measured by a BCA protein assay on the following day.
Supplemental Figure 4 - Cordie
Supplemental Figure 4.Attachment and proliferation of fibroblasts on nanofibrous substrates. A) Attachment of WA01-H2B-mCherry fibroblasts at 24 hours. Cells were counted by flow cytometry after dissociation from substrates. B) Proliferation of fibroblasts over 72 hours. Proliferation Index is the ratio of cell counts after 24 to cell counts after 72 hours.
Supplemental Figure 5 - Cordie
Supplemental Figure 5. Attachment and proliferation of pluripotent stem cells on nanofibrous substrates. A) Attachment of WA09hESCs at 24 hours. Cells were counted by flow cytometry after dissociation from substrates. B) Proliferation of fibroblasts over 72 hours. Proliferation Index is the ratio of cell counts after 24 to cell counts after 72 hours. C) Growth rate comparison of parental WA01 and daughter WA01-H2B-mCherry cell lines.
Supplemental Figure 6 - Cordie
Supplemental Figure 6.Kinetics of reprogramming on nanofibrous substrates.Fraction of reprogramming population belonging to each of three cellular identities (see also Fig. 2A-B) over a 22 day time course of reprogramming on nanofibrous substrates.All experiments were conducted in E7 media with hydrocortisone and doxycycline.
Supplemental Figure 7 - Cordie
Supplemental Figure 7.Nuclear morphology and histone acetylation on nanofibrous substrates.
A) Effects of nanofibrous substrates on nuclear size of C1.2 fibroblast cells during normal growth (Basal, E7 media, light blue) and during reprogramming (E7 media with hydrocortisone and doxycycline, dark blue). After seven days of treatment, cells were fixed and stained nuclei were analyzed. Error bars denote 95% confidence intervals. Asterisks indicate p-values from an unpaired student’s t-test(**p<0.001, ***p<10-5).B) Representative histogram from flow cytometric analysis of histone acetylation (AcH3).C) Mean AcH3 levels at day 7 weakly correlated with nuclear shape index.
Supplemental Figure 8 - Cordie
Supplemental Figure 8.TGF-β inhibits iPSC formation on nanofibrous substrates. Change in the iPSC reprogramming population on various substrates when media is supplemented with TGF-β.
Supplemental Figure 9 - Cordie
Supplemental Figure 9. Partial least squares regression of biophysical and biochemical cues and many cellular responses. A) Contribution of each cue to principal components in the two-component model as described in Fig. 6. B) Predictability of the full PLSR model describing 8 responsive variables and 5 principal components. C) Root mean square error in predictive calculation. Number of components was picked to reduce probability of overfitting. D) Cumulative sum of variance explained by increasing number of components in the full model.
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