Artificial niche microarrays for probing single-stem-cell fate in high throughput

Transcription

1 Nature Methods Artificial niche microarrays for probing single-stem-cell fate in high throughput Samy Gobaa, Sylke Hoehnel, Marta Roccio, Andrea Negro, Stefan Kobel & Matthias P Lutolf Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Photograph of a silicon stamp produced by etching a silicon wafer with a photolithography process. Example of production of hydrogel microwell array in a multiwell plate Confocal image of an individual ProteinA-modified hydrogel microwell. Coefficient of variation measured for both surface and differentiation indexes as a function of the initial cell density in the microwells. Immunostaining of MSCs cultured for 11 days in media inducing either proliferation, osteogenic or adipogenic differentiation. Supplementary Figure 6 Quantitative PCR performed on hmscs cultured for 11 days in media inducing either proliferation (P), osteogenic (O) or adipogenic (A) differentiation. Supplementary Figure 7 Supplementary Figure 8 Supplementary Figure 9 Supplementary Figure 10 Supplementary Figure 11 Supplementary Table 1 Supplementary Table 2 Combined effects of N-cadherin and initial cell density per microwell on the proliferation of hmscs. Effect of inhibition of N-cadherin interaction on adipogenic differentiation at variable cell numbers per microwell. NSC differentiation on poly(l-lysine)-coated plastic dishes. NSC differentiation on PEG hydrogel microwell arrays. Four printing profiles used to generate artificial niche microarrays. Proteins used for NSC niche screening assay. Primers for quantitative PCR

2 Supplementary Fig. 1 Supplementary Fig. 1: Photograph of a silicon stamp produced by etching a silicon wafer with a photolithography process. One stamp embeds 2016 microwell organized in seven 12X24 fields. Each micropillar has a diameter of 450 µm and a height of approximately 100 µm.

3 Supplementary Fig. 2: Supplementary Fig. 2: Example of production of hydrogel microwell array in a multiwell plate (here: 12-well plate). Photograph of (a) the microfabricated holder used for aligning the mini-stamps (b) in the robotic spotter. After protein printing, the mini-stamps can be pressed against (c) the hydrogel layer at the bottom of the 12-well plate.

4 Supplementary Fig. 3 Supplementary Fig. 3: Confocal image of an individual ProteinA-modified hydrogel microwell. The gel is stained with Alexa-555 labeled Fc-fragment to visualize the geometry of the microwell.

5 Supplementary Fig. 4 Supplementary Fig. 4: Coefficient of variation measured for both surface and differentiation indexes as a function of the initial cell density in the microwells. Measurements were obtained under adipogenic differentiation conditions. In the population of one cell per microwell at day 0, a CV of 214 % was measured, in contrast to the population of twelve cells at day 0, which resulted in a CV-decrease by a factor of three (66 %).

6 Supplementary Fig. 5 Supplementary Fig. 5: Immunostaining of MSCs cultured for 11 days in media inducing either proliferation, osteogenic or adipogenic differentiation. Nuclei are stained with Dapi and N-Cadherin by a primary mouse anti-ncadherin (1:400) and a secondary anti-mouse Alexa488 (1:400).

7 Supplementary Fig. 6 Supplementary Fig. 6: Quantitative PCR performed on hmscs cultured for 11 days in media inducing either proliferation (P), osteogenic (O) or adipogenic (A) differentiation. Primers (Supplementary Table 2) were chosen to reveal the variations in the expression of GAPDH, LPL and N-Cadherin. Error bars represent the standard deviation.

8 Supplementary Fig. 7 Supplementary Fig. 7: Combined effects of N-Cadherin and initial cell density per microwell on the proliferation of hmscs.

9 Supplementary Fig. 8 Supplementary Fig. 8: Effect of inhibition of N-Cadherin interaction on adipogenic differentiation at variable cell numbers per microwell. N-Cadherin activity was blocked by adding anti-n-cadherin antibody (Clone CG4, Sigma) to the culture medium.

10 Supplementary Fig. 9 Supplementary Fig. 9: NSC differentiation on poly(l-lysine)-coated plastic dishes. Induction of differentiation was obtained by reducing EGF concentrations to 0.2ng/ml for 10 days. Scale bar = 50µm.

11 Supplementary Fig.10 Supplementary Fig. 10: NSC differentiation on PEG hydrogel microwell arrays. Cells were seeded at clonal densities (104 per array) and differentiated induced by culture in reducing EGF concentration (neurons, oligodendrocytes) or by adding serum and retinoic acid (astrocytes). Scale bar = 100µm

12 Supplementary Fig. 11

13 Supplementary Fig. 11: Four printing profiles used to generate artificial niche microarrays. (A) Array design of the overlapping BSA gradients. (B) A full randomization pattern where the organization of the microwells are random inside each field. (C) Organization of the microwells for immobilization of eight dilutions of FN Three randomization were spotted twice per array. (D) The array design used for producing the cellular arrays used to immobilize the proteins indicated in Supplementary Table 1.

14 Supplementary Table 1: Proteins used for NSC niche screening assay. Supplementary Table 2: Primers for quantitative PCR Gene Type Primer sequence N-Cadherin Forward 5 -GGCAGAAGAGAGACTGGGTC-3 Reverse 5 -GAGGCTGGTCAGCTCCTGGC-3 LPL Forward 5 -GAGATTTCTCTGTATGGCACC-3 Reverse 5 -CTGCAAATGAGACACTTTCTC-3 GAPDH Forward 5 -GAAGGTGAAGGTCGGAGTC-3 Reverse 5 -GAAGATGGTGATGGGATTTC-3