Artificial cells drive neural differentiation

Stimuli-responsive artificial cells synthesize and controllably release therapeutics for neural differentiation.

. Details of the optimization of the E. coli S12 reaction for physiological conditions. In vitro transcription-translation reactions were at 30 °C for 12 h. Bar graphs show final time points. Fluorescence experiments monitored the emission arising from the expression of superfolder GFP from construct DT035A (same as fig. S1) (A) The effect of different transcriptional promoter strengths. All N-terminal sequences were identical except for the promoter region. Details on the promoters are in the Methods and Table S1. (B) Effect of supplementary solutions on the synthesis of sfGFP, Mg 2+ -glutamate/K + -glutamate denoted as Mg 2+ /K + . (C) Effect of potential contaminants of the optimized S12 reaction. PBS, DMEM, alanine (300 mM in nuclease-free water) were added as 10% (v/v) of the reaction volume. No negative effect was detected compared to the positive control, which contained nuclease-free deionized water. (D) Left: Effect of maltose on the end point (10 h). Right: Effect of maltose on early transcription-translation. (E) Effect of the macromolecular crowding agent PEG 8000 on transcription-translation. Values above 2% (w/v) PEG 8000 gave higher yield; however, due to the increased osmolality of the E. coli S12 reaction, we chose 2% (w/v) for further experiments. (F) Cartoon representations of each construct. m.BDNF indicates mature BDNF (DT033A), pr.MW is a hybrid transcriptional promoter (BBa_J23106 and BBa_J23117, DT034A), and pdt is a Mesoplasma florum protein degradation tag (62). (G) Left: Western blot analysis of BDNF, LuxR, and PFO expression for the optimization of plasmid concentration. Immunoblotting was against the N-terminal FLAG-tag. Strong transcriptional promoters were screened for BDNF. Hybrid weak promoter of LuxR allowed for the synthesis of PFO by in vitro transcription-translation upon induction with 10 µM 3OC6 HSL. To ensure that the limited resources of the artificial cell were primarily directed toward the synthesis of BDNF (63), strong and weak promoters were selected for the expression of BDNF and LuxR, respectively. Right: Optimal plasmid concentrations (green) for protein production were 20 nM for the BDNF plasmid (DT033A) and 10 nM for the LuxR-PFO plasmid (DT034A). Induction of PFO expression was after 45 min incubation at 30 °C.

fig. S3. Overview and validation of vesicle formation by the inverted emulsion method. (A)
A mineral oil (light oil, ρ = 0.84 g/mL) solution was prepared to dissolve the lipid components. Water-in-oil (inverted) emulsions were prepared with 2% (v/v) aqueous solution in the lipid-mineral oil phase. For example, 10 µL of inner solution was pipetted into 500 µL lipid/mineral oil solution. Subsequently, the 1.5 mL tube containing the mixture was rapidly drawn over a rack to form inverted emulsions (see Methods for more details). A mild centrifugal force was applied to form vesicles taking advantage of the density difference between outer (ρalanine = 1.42 g/mL) and inner solutions (ρsucrose = 1.59 g/mL). (B) Pictures illustrating the process of forming vesicles. Photo Credit: Ömer Duhan Toparlak, University of Trento. (C) Microscopy images of sucrose containing vesicles generated by the inverted emulsion technique described in panel (A). The inner solution composition was 280 mM sucrose and 500 µM HPTS. An equiosmolar alanine solution (i.e. 300 mM) or DPBS (without Mg 2+ and Ca 2+ ) was used as outer solution, to take advantage of density difference between inside and outside the vesicles. Identical results were obtained with the E. coli S12 extract. To enhance the density difference and the efficiency of the cell-free reaction, the inner solution was further supplemented with 20 mM of sucrose and 6 mM of maltose ( fig. S1C and S2D). Scale bar indicates 100 µm. (D) Flow cytometry analysis of artificial cells generated by inverted emulsion technique. The inner solution for artificial cells contained osmotically optimized E. coli S12 reaction and 10 µM AlexaFluor488 labeled 10 kDa dextran. The instrument was calibrated with polystyrene beads of known size. Upon calibration, the giant vesicle fraction was selected for uniform particle distribution and reliable characterization of the artificial cell population.

fig. S4. Supporting data for artificial cell functionality with neural stem cells. (A) Statistical analysis of MAP2
and βIII-Tubulin immunostaining. Overlapping signals were used to assess the maturation levels and signal specificity. Data indicate the mean of three different fields with ± SD. The statistical test was student's t-test (unpaired, two-tailed), where p < 0.05 was considered significant. (B) Representative microscopy images of immunostaining of mNS cells at 19 days in vitro, after artificial cell treatment, antibodies were against cleaved Caspase-3 (red) to assess apoptosis and βIII-Tubulin (green) to assess the success of differentiation to appreciate non-overlapping cleaved Caspase-3 signals. (C) Statistical analysis of overall cell population for cleaved Caspase-3. Data indicate mean of three independent experiments (n = 3) and error bars indicate ± SEM. The statistical test was student's t-test (unpaired, two-tailed), where p < 0.05 was considered significant. 'n.s.' stands for 'not significant'. (D) Representative immunostaining images of mNS cells at 19 days in vitro, after artificial cell treatment. a-Glial Fibrillary Acidic Protein (GFAP, red) antibody was used to assess the glial cells i.e. astrocyte population during differentiation and maturation with artificial cells. (E) Statistical analysis of overall cell population for GFAP. Data indicate the mean of two different fields with ± SD. The statistical test was student's ttest (unpaired, two-tailed), where p < 0.05 was considered significant. (F) Raw data for the differentiation of mNS cells into neurons. An artificial cell formula encapsulating commercial BDNF and releasing upon sensing 3OC6 HSL was used to assess the quality of neural differentiation. "Com. BDNF" stands for Commercial BDNF (Peprotech) All data points are biological replicates. This dataset was used to generate  Fig. 2 and Fig. 3. (B) Stability of artificial cells with 10% (v/v) FBS at 24 h. Artificial cells that were not in contact with eukaryotic cells are denoted as a control group and used as a reference for maximum FITC+ events, in both FBS and Transwell tests. In this group, the artificial cells were separately incubated at 37 °C in PBS for 24 h and then briefly washed with eukaryotic cells and isolated immediately, in order to match the same culture environment as in test group. In the presence of FBS, the population of artificial cells showed a ca. 7% decrease within 24 h, taking the control vesicles group as 100%. However, this effect was not observed in the absence of FBS. Presumably the rapid increase in osmolality, unknown components of the bovine serum, and an increase in eukaryotic cell-derived events due to cell growth are the three contributing factors for the loss of artificial cell events.

fig. S14. Toxicity of artificial cells to HEK293T cells. (A)
MTT assay results following co-incubation with recombinantly expressed and purified PFO. Inhibitory concentration (IC50) of recombinant PFO was found to be ca. 3.7 nM for HEK293T cells. This value is likely to be a higher concentration of PFO than artificial cells can produce and release. This control was used as a reference. These data combined with the data in fig. S8E were consistent with intravesicularly produced and/or leaked amounts of PFO roughly equal to or less than 4 nM. (B) MTT assay results following co-incubation with no-DNA-containing artificial cells at days 1 and 7. No significant cytotoxic effects were observed with HEK293T cells. The erratic readings are due to cell growth and seeding differentials, as similar growth deficiencies were also observed in non-treated wells. A single dose of artificial cells is determined as the final amount used to generate Fig. 2. (C) MTT assay results following co-incubation with no-DNA-containing cell-free extract at days 1 and 7. Within 24 h, the cell-free reaction dose used with HEK293T cells for the assessment of the functionality of cell-free synthesized BDNF (0.25 µL) did not show any cytotoxic effect. Following 1-week incubation, the cell-free reaction began to exert a cytotoxic effect. In these assays, the complete cell death was achieved by adding Triton X-100 at a final concentration of 0.5 % (v/v), which was the negative control. The positive control was the no-treatment group. The positive control was considered 100% cell viability, whereas the negative control was used as 0% cell viability. The data were fitted to sigmoidal, where X values were considered as concentration, to generate IC50 values on GraphPad Prism 7. Cultures treated with commercial BDNF and PBS were considered as 0% and 100%, respectively. All experiments were performed as independent biological replicates, n = 3. Data show mean, and error bars represent ± SEM. Statistical tests were student's t-test (unpaired, two-tailed).
fig. S16. Quantification of cell-free synthesized BDNF. 6xHis-sfGFP was recombinantly expressed in E. coli BL21 (DE3) and purified with a Ni 2+ -NTA column. Known amounts of 6xHis-sfGFP, determined by the extinction coefficient, were used to generate stock solutions. The western blotting of FLAG-tagged BDNF and FLAG-tagged sfGFP was performed with serial dilutions (1:1 dilutions) to generate a standard curve. The linear fits of blot quantification were used to estimate the amount of BDNF and sfGFP produced from the in vitro transcriptiontranslation reaction. The data were then compared to known amounts of purified 6xHis-tagged sfGFP.    TCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGA  GGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAA  CTGCCGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTC  GTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGT  GCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAA  TTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACG  GCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGC  CGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGC  AGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGC  TGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACG  CGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAA  CTGTACAAATAATAAcgactcaggctgctacgcctgtgtactggaaaacaaaaccaaaacc  caaaaaacaaaaaACTGAGCCCATTGGTATCGTGGAAGGACTCgatcaaaaaaaaaaaaaa  aaaaaaaaaaaaaaaaCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTT  TTG  DT041A CMV-TrkB   CGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA  GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC  CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG  ACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATC  AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG  GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTA  GTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGT  TTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA  CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGC  GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCA  CTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTTGGTACCA  TGTCGCCCTGGCTGAAGTGGCATGGACCCGCCATGGCGCGGCTCTGGGGCTTATGCCTGCT  GGTCTTGGGCTTCTGGAGGGCCTCTCTCGCCTGCCCGACGTCCTGCAAATGCAGTTCCGCT  AGGATTTGGTGTACTGAGCCTTCTCCAGGCATCGTGGCATTCCCGAGGTTGGAACCTAACA  GCGTTGACCCGGAGAACATCACGGAAATTCTCATTGCAAACCAGAAAAGGCTAGAAATCAT  CAATGAAGATGACGTTGAAGCTTACGTGGGGCTGAGAAACCTTACAATTGTGGATTCCGGC  TTAAAGTTTGTGGCTTACAAAGCGTTTCTGAAAAACAGCAACCTGCGGCACATAAATTTCA  CACGAAACAAGCTGACGAGTTTGTCCAGGAGACATTTCCGCCACCTTGACTTGTCTGACCT  GATCCTGACGGGTAATCCGTTCACGTGCTCCTGCGACATCATGTGGCTCAAGACTCTCCAG  GAGACTAAATCCAGCCCCGACACTCAGGATTTGTACTGCCTCAATGAGAGCAGCAAGAACA   TGCCCCTGGCGAACCTGCAGATACCCAATTGTGGTCTGCCATCTGCACGTCTGGCTGCTCC  TAACCTCACCGTGGAGGAAGGAAAGTCTGTGACCCTTTCCTGCAGTGTGGGGGGTGACCCA  CTCCCCACCTTGTACTGGGACGTTGGGAATTTGGTTTCCAAGCACATGAATGAAACAAGCC  ACACACAGGGCTCCTTAAGGATAACGAACATTTCATCTGATGACAGTGGAAAGCAAATCTC  TTGTGTGGCAGAAAACCTTGTAGGAGAAGATCAAGATTCTGTGAACCTCACTGTGCATTTT  GCGCCAACTATCACGTTTCTCGAGTCTCCAACCTCAGATCACCACTGGTGCATTCCATTCA  CTGTGAGAGGCAACCCCAAGCCTGCGCTTCAGTGGTTCTACAATGGGGCCATACTGAATGA  GTCCAAGTACATCTGTACTAAGATCCACGTCACCAATCACACGGAGTACCATGGCTGCCTC  CAGCTGGATAACCCCACTCATATGAATAACGGAGACTACACCCTGATGGCCAAGAACGAGT  ATGGGAAGGATGAGAGACAGATCTCCGCTCACTTCATGGGCCGGCCTGGAGTCGACTACGA  GACAAACCCAAATTACCCTGAAGTCCTCTATGAAGACTGGACCACGCCAACTGACATTGGG  GATACTACGAACAAAAGTAATGAAATCCCCTCCACGGATGTTGCTGACCAAAGCAATCGGG  AGCATCTCTCGGTCTATGCCGTGGTGGTGATTGCATCTGTGGTGGGATTCTGCCTGCTGGT  GATGTTGCTCCTGCTCAAGTTGGCGAGACATTCCAAGTTTGGCATGAAAGGCCCAGCTTCG  GTCATCAGCAACGACGATGACTCTGCCAGCCCCCTCCACCACATCTCCAATGGGAGTAACA  CTCCATCTTCTTCGGAGGGCGGTCCCGACGCTGTCATTATTGGAATGACCAAGATTCCTGT  TATTGAAAACCCCCAGTACTTTGGCATCACCAACAGTCAGCTCAAGCCAGACACATTTGTT  CAGCATATCAAGAGACACAACATCGTTCTGAAGAGGGAACTTGGGGAAGGAGCCTTCGGGA  AAGTTTTCCTTGCCGAGTGCTACAACCTCTGCCCAGAGCAGGATAAGATCCTGGTGGCTGT  GAAGACGCTGAAGGACGCCAGCGACAATGCACGCAAGGACTTTCATCGGGAAGCTGAGCTG  CTGACCAACCTCCAGCACGAGCACATTGTCAAGTTCTACGGTGTCTGTGTGGAGGGCGACC  CACTCATCATGGTCTTTGAGTACATGAAGCACGGGGACCTCAACAAGTTCCTTAGGGCACA  CGGGCCCGACGCAGTGCTGATGGCAGAGGGTAACCCGCCCACAGAGCTGACGCAGTCGCAG  ATGCTGCACATCGCTCAGCAAATCGCAGCAGGTATGGTCTACCTGGCGTCCCAACACTTTG  TGCACCGTGACCTGGCCACCCGGAACTGCCTGGTGGGAGAGAACCTGCTGGTGAAAATTGG  GGACTTTGGGATGTCCCGAGATGTGTACAGCACCGACTACTATCGGGTCGGTGGCCACACA  ATGTTGCCCATCCGATGGATGCCTCCAGAGAGCATCATGTACAGGAAATTCACCACCGAGA  GCGACGTCTGGAGCCTGGGCGTTGTGTTGTGGGAGATCTTCACCTACGGCAAGCAGCCCTG  GTATCAGCTATCGAACAATGAGGTGATAGAGTGCATCACCCAGGGAAGAGTCCTTCAGCGG  CCTCGAACGTGTCCCCAGGAGGTGTATGAGCTCATGCTTGGATGCTGGCAGCGGGAACCAC  ACACCCGGAAGAACATCAAGAGCATCCACACCCTCCTTCAGAACTTGGCCAAGGCATCTCC  CGTCTACCTGGATATCCTAGGCTAGTAGTCTAGAGGGCCCTATTCTATAGTGTCACCTAAA  TGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGC  CCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA  ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG  GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGC  TCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCT  GTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGC  C *From DT033A to DT040A, promoter sequences are in blue, spacer or polyA sequences are in lowercase, ribosome binding sites are in italic letters, FLAG-tag sequences are in green, the first and the last codons of the protein sequences are in bold, protein degradation tag sequences are in cyan, FRET-pair binding sequences (to monitor mRNA levels in vitro) are in purple, and terminator sequences are highlighted in red. For DT041A, the Cytomegalovirus (CMV) enhancer sequence is indicated in green, the CMV promoter sequence is indicated in blue, the first and the last codons of murine TrkB sequence are in bold.