Synthesis and characterization of MnO 2 nanosheet and GO

MnO 2 nanosheets were synthesized based on a previous protocol with minor modifications54. Briefly, 2.2 g Tetramethyl ammonium pentahydrate (TMAOH·5H 2 O, Alfa Aesar) was first dissolved in 20 ml 3% wt. H 2 O 2 (Sigma-Aldrich) by vortexing (concentration of TMAOH is 0.6 M). In parallel, 0.594 g MnCl 2 ·4H 2 O (Sigma-Aldrich) was dissolved in 10 ml de-ionized water (Concentration of MnCl 2 is 0.3 M) through sonication. Then the TMAOH dissolved in H 2 O 2 solution was rapidly added into MnCl 2 solution within 10 s with fast stirring at 1200 r.p.m. (round per minute). Please note that gas will be generated and rapid increase of solution volume will be observed. The solution was continued to be stirred at 600 r.p.m. overnight and centrifuged at 2000×g for 5 min to obtain the bulk δ-MnO 2 . After washing with water for 3 times and ethanol for 2 times through shaking and centrifuge, bulk MnO 2 was dried in the oven under ambient conditions for 12 h. After adding 100 mg into 10 ml de-ionized water, the solution was extensively sonicated in the Sonics bath sonicator for 10 h. Lastly, the solutions were centrifuged at 8801×g for 10 min to get rid of the aggregations and un-exfoliated products. The black solution was measured with concentration by evaporating water in the solution. MnO 2 nanosheet was diluted to 10 μg per ml for TEM (80 kv on a Philips CM12 with an AMT digital camera model XR111) and Ultra-Stem imaging. For X-ray photoluminescence spectroscopy (Thermo Scientific ESCALAB 250 Xi with a base pressure <1 × 10–9), MnO 2 nanosheet solution (100 μg per ml) was drop-casted onto a silicon substrate and dried in the vacuum. An Al-Kα monochromated X-ray source was used to obtain the core level spectra, and the instrumental broadening was around 0.5 eV. The hydrodynamic size and zeta potential of MnO 2 nanosheets in aqueous solution were measured by a ZS (Nano Zetasizer) dynamic light scattering instrument (Malvern Instruments, Malvern, UK), with the temperature set at 25 °C and a detection angle of 90 degrees. UV-Vis absorption spectrum of MnO 2 nanosheet solution was measured by a Varian Cary 50 spectrophotometer using a quartz cuvette.

Graphene oxide was synthesized based on our previous publications55,56. 1.0 gram of Graphite (Bay Carbon) was preoxidized in the mixture of sulfuric acid (Sigma-Aldrich, 98%), phosphor oxide (Sigma-Aldrich) and potassium persulfate (Sigma-Aldrich) at 80 degrees for overnight. Then the pre-oxidized graphite was washed with water, dried and reacted with sulfuric acid and potassium permanganate through a 3-step process. After quenching with H 2 O 2 , a shining gold solution appear, and the graphite oxide was purified with 10% HCl solution (Sigma-Aldrich) and water extensively. Lastly, graphite oxide was exfoliated into graphene oxide by tip sonication (Branson). Multi-layered graphene oxide was centrifuged down at 16,639×g for 45 min, and the final suspension of single or few layered graphene oxide was obtained.

Measurement of protein absorption by MnO 2 nanosheet

Into the solutions of ECM protein (laminin protein from Sigma-Aldrich, stock concentration of 200 μg per ml, 0.5 ml, PBS is from Thermo Fisher), 10 μl of MnO 2 nanosheet aqueous solution (3 mg per ml) was added, or a piece of etched glass (first treated by pirahana solution for 1 h, then oxygen plasma treated for 1 min, followed by polylysine (1 mg per ml) coating for 4 h) or a polymer scaffold (Polycaprolactone nanofiber scaffold23, 1 mg) was inserted. The solutions turned brown immediately, and then they were continued to incubate under 37 °C for 1 h. To remove the MnO 2 nanosheet with absorbed proteins, the solution was centrifuged for 3 times at 8801×g for 10 min, and precipitates were removed each time until there are no visible precipitates anymore. The solution should be transparent at this moment. 0.1 ml supernatant solution was transferred into a 96-well plate, and BCA (bicinchoninic acid assay, Thermo Fisher, A53226) was used to quantify the percentage of protein absorbed on nanosheets by subtracting the total amount of proteins remaining in the control group to the protein remained in the experimental groups. The assay was conducted strictly following the protocols from Thermo Fisher and absorption at 570 nm was used to quantify the protein amount for each group. These experiments were replicated for 3 times, and the values were normalized to the glass control. The amount of laminin absorbed on MnO 2 nanosheet was calculated by subtracting the laminin concentration after MnO 2 nanosheet absorption from the original concentration. The percentage of laminin absorption was calculated by dividing the amount of laminin absorbed by the original laminin concentration. BCA protein assay was repeated 3 times experimentally to get error bars shown in Fig. 1. Data are mean ± s.d., n = 3, **P < 0.01 by one-way ANOVA with Tukey post-hoc test.

In vitro biodegradation of MnO 2 hybrid nanoscaffold

We first studied the in vitro degradation of MnO 2 nanosheet in physiological conditions, different PBS solutions with varying concentrations of vitamin C (10 μg per ml, 50 μg per ml, 100 μg per ml, 200 μg per ml, 500 μg per ml) were prepared. Then 10 μl of 3 mg per ml MnO 2 nanosheet solution was added into 3 ml of vitamin C solution, and the UV-Vis spectrum of the solution was recorded every two minutes. The percentage of nanosheet remaining was normalized to the absorption (at 385 nm) of a control group without any vitamin C added. To study the degradation of thin layered nanoscaffold, we drop-casted 100 μL of MnO 2 nanosheet solution (1.0 g per ml) into the wells of 24 well plate treated with oxygen plasma. After vacuum drying for 3 h, a homogeneous, yellow and transparent film formed. Then the wells were coated with laminin (Thermo Fisher, Catlog No.: 23017015) and seeded with human neural stem cells at a cell density of 80k per well. The cells were cultured in standard differentiation media (w/o bFGF and no exogenous compounds) for different periods of time (1 day, 3 day, 7 day, 12 day, 17 day, and 22 day). The cells were then fully detached using acutase for 10 min at 37 °C. Followed by washing with PBS and de-ionized water, 24-well plate was vacuum dried. Based on the absorption of MnO 2 nanosheet at 385 nm, the degradation percentage was quantified by subtracting background (empty well) and normalize to the well without culturing the cells.

To study the degradation of thick layered nanoscaffold, a 3-layer, cell-MnO 2 nanoscaffold-cell sandwiched structure that mimic tissue structures was formed through centrifugation at 130×g in a 15 mL Eppendorf centrifuge tubes. A similar structure of GO nanoscaffold-cell construct was formed using the same protocols as a control. The first layer contains 1 million iPSC-NSCs. The second layer is composed of 1.0 mg of MnO 2 nanosheet or graphene oxide. The third layer (Top layer) was centrifuged down from another 1 million iPSC-NSCs. Degradation of the scaffolds was monitored by the volume change and thickness change of scaffold on a weekly base. Based on the assumption that the scaffold has identical radius and areas, the percentage of scaffold volume was normalized to the thickness that was measured on Day 1.

To demonstrate cell-seeded nanoscaffold can fast degrade under biocompatible redox conditions, an aqueous solution of 0.3 mg per ml MnO 2 nanosheet was filtered through a cellulose membrane, then a layer of dye-labeled (food dye) cells was formed on the MnO 2 nanosheet assembled substrate using a tri-circular PDMS (Polydimethylsiloxane, Dow Corning®) chamber. After the addition of 2.0 mg per ml ascorbic acid (Sigma-Aldrich) for 5 min, most of the dark color of MnO 2 nanosheet disappeared, and a layer pink colored cell layer was formed.

Tunable biodegradation of MnO 2 nanoscaffolds

To show the tunable biodegradability of MnO 2 nanoscaffolds, we controlled the geometrical and chemical structures of MnO 2 nanoscaffold (Supplementary Fig. 10) which includes: (i) thickness [0.2 H vs. 1 H (H = 0.4 mm), shown in a and b, respectively, which is achieved by filtrating different concentrations (0.6 mg per ml and 3.0 mg per ml, respectively) of MnO 2 nanosheet solution while keeping solution volume (1.0 ml) and filtrating area (1075 mm2) constant]; (ii) Height to surface area ratio (from 0.4 mm:1075 mm 2 in Supplementary Fig. 10b to 4 mm:107.5 mm2 in Supplementary Fig. 10c), which was achieved by filtrating same amount of MnO 2 nanosheets (1.0 ml of 3.0 mg per ml) but reduced the filtrating area by 10 times; (iii) protein amount in the scaffold (MnO 2 nanosheets absorbed with 1.0 mg per ml vs. with 10 mg per ml bovine serum protein and then vacuum filtered). Degradation profile of different scaffolds obtained by measuring time-dependent manganese concentrations in the solution using Inductively coupled plasma mass spectrometry (ICP-MS, Fisons Instruments PlasmaQuad 2+). Each sample was measured 3 times to obtain error bars and standard deviation. To control scaffold degradation without any additional bioreductants, different cell densities (0, 0.1, 0.5, 1, and 5 million cells per well in a 24 well plate) were seeded onto MnO 2 nanoscaffold, and the complete degradation time was monitored by the full disappearance of dark color. 0.5 ml media was changed every two days. Each experiment was repeated twice. Results are summarized in Supplementary Fig. 10m.

Fabrication and characterization of MnO 2 hybrid nanoscaffold

To fabricate the MnO 2 hybrid nanoscaffolds, 10 ml of MnO 2 nanosheet solution at a specific concentration was filtered through a cellulose filter paper (pore size = 20 nm) under the vacuum condition. Then the filter paper was taken out and cut into sizes and shapes of choice. To transfer into a transparent glass substrate, cleaned glass was first treated with oxygen plasma, then the MnO 2 nanosheet deposited on filter paper was wetted with de-ionized water and pressed against the glass. A 2.0 kg per cm2 pressure was placed on top of the filter paper for 8–12 h, and glass attached with MnO 2 nanosheet was detached from the weight. To remove the cellulose attached with nanoscaffold, the substrate was incubated in acetone for 0.5 h and then briefly washed in methanol for 1 h. The transparency of nanoscaffold can be easily tuned by using different concentrations of MnO 2 nanosheet solution. The 3 concentrations used in Supplementary Fig. 3 are 50 μg per ml, 100 μg per ml and 200 μg per ml. For cellular studies, the concentration of 200 μg per ml MnO 2 nanosheet solution was used throughout the study. Graphene oxide assembled scaffold was fabricated using an identical protocol with a graphene oxide aqueous solution of 200 μg per ml. FESEM (Field Emission Scanning Electron Microscopy, Zeiss with Oxford EDS) was used to characterize the nanoscaffold.

To form MnO 2 laminin hybrid nanoscaffold, 400 μL of MnO 2 nanosheet aqueous solution (2 mg per ml) was quickly added to 100 μL of laminin solution (1.0 mg per ml, PBS, PH = 7.4). Then the laminin conjugated MnO 2 nanosheet was centrifuged and re-suspended in 10 ml de-ionized water (MnO 2 nanosheet concentration was 80 μg per ml). After vacuum filtration, the cellulose filter paper or PCL fiber was cut into size and shape of interest for the following cell studies.

HiPSC-NSC culture

Human iPSC-NPCs were derived from human iPSCs (WT126 clone 8; and WT33 clone 1)57. iPSC-NPCs were expanded in a proliferation media containing DMEM/F12 with Glutamax (Invitrogen), B27-supplement (Invitrogen), N2 (Stem Cells), and 20 ng per mL FGF2 (Invitrogen). Tissue culture vessels were treated with Matrigel (Corning) 1:200 dilution with DMEM (Invitrogen) at 37 °C for 1 h. To initiate the neuronal differentiation process, bFGF was removed. Fresh media was exchanged every other day. iPSC-NSCs with passage 8–11 were used in all our transplantation and in vitro studies.

Differentiation of iPSC-NSC on MnO 2 nanoscaffold

The viabilities of iPSC-NSCs and human neural progenitor cells (hNPC, Supplementary Methods) cultured on MnO 2 nanoscaffold were measured by presto blue cell viability assay (Thermo Fisher, Catalog No.: A13261, 10% volume ratio as compared to cell media). Into 24-well plates, laminin (10 μg/ml) was first coated onto the glass (control), graphene oxide assembled scaffold (positive control) and MnO 2 nanoscaffold at a concentration of 20 μg per ml (media volume: 1.0 ml per well), for 4 h. Then the iPSC-NSC was seeded into each well at a cell density of 20 k in growth media (bFGF added, 20 ng per ml). After the cells were cultured for 48 h, cell viabilities cultured on different substrates were quantified using fluorescence (excitation at 570 nm and emission at 590 nm) presto blue assay and normalized to the control group (glass). For the neurite length analysis, neurites on each substrate were first automatically traced and the lengths were automatically measured by NeuronJ in ImageJ software. The values are all averaged from 9–12 measurements in the representative immunostaining images58. Here is a summary of the average neurite lengths with standard deviation obtained from software: Glass control, 5.2 ± 2.3 μm; MnO 2 nanoscaffold, 59.3 ± 19.8 μm; MnO 2 laminin nanoscaffold, 84.5 ± 26.5 μm.

For the differentiation of iPSC-NSC on glass substrates, MnO 2 nanoscaffold, graphene oxide assembled scaffold (GO nanoscaffold) and glass were first sterilized in the ultraviolet (UV) lamp for 5 min and then coated with laminin solution (10 μg per ml) for 4 h. The substrates were placed in 24-well plates, and iPSC-NSCs were seeded into the wells at a cell density of 60 k cells per well. The cells proliferated for 24 h, and the media was changed to differentiation media without bFGF. To observe the stem cell proliferation and attachment onto the substrate, the cells were imaged in the optical microscope (Nikon Eclipse Ti-E microscope). After 6-days’ differentiation, the cells were fixed and immunostained with nuclei (Hoechst, Thermo Fisher, catalog number: 33346, 1:100 dilution, 0.2 mM) and neuronal marker (TuJ1, Cell Signaling, catalog number: 4466, 1:500 dilution). To quantify the neuronal markers (TuJ1) and astrocyte markers (GFAP), qRT-PCR was conducted by using GAPDH mRNA as a control (Supplementary Table 2).

Fabrication of MnO 2 laminin hybrid nanoscaffold

MnO 2 laminin hybrid nanoscaffold can be facilely fabricated by adding 10 μL of MnO 2 nanosheet aqueous solution (3 mg per ml) into 100 μL laminin solution (1 mg per ml), and the MnO 2 nanosheet will be assembled within 5 s. To fabricate larger scale MnO 2 laminin hybrid nanoscaffold, 100 μL of MnO 2 nanosheet aqueous solution (3 mg per ml) was added into 500 μL laminin solution (1 mg per ml) and then vacuum filtered on a cellulose paper as described above. The structure of MnO 2 laminin hybrid nanoscaffold was then analyzed in FESEM. To fabricate cell encapsulated MnO 2 laminin-nanoscaffold, 1 million iPSC-NSCs were centrifuged down and re-dispersed in 25 μL laminin PBS solution. Different amount (0, 0.3, 1.5, 3, 15, and 30 μL) of MnO 2 nanosheet solution (3 mg per ml) was injected into the cell laminin solution, and a iPSC-NSC encapsulated pellet was spontaneously formed after one hour. To investigate the interaction between MnO 2 and encapsulated iPSC-NSCs, the medium was removed and the neurons were fixed in Formalin solution (Sigma-Aldrich) followed by two PBS washes. The biological samples (prepared under 15 μL MnO 2 condition) were then dehydrated to eliminate water through a series of ethanol dehydration process by replacing PBS with 50% ethanol/water, 70% ethanol/water, 85% ethanol/water, 95% ethanol/water, and absolute ethanol twice for 10 min each in succession. The biological samples were then stored in absolute ethanol before transferring to critical point dryer to eliminate traces of ethanol. Then 20 nm of gold was sputter coated onto the surface of biological samples after drying. FESEM was then used for micrograph acquisition.

For the differentiation of iPSC-NSC on substrates, glass, MnO 2 nanoscaffold and MnO 2 laminin hybrid nanoscaffold were first sterilized in the UV lamp for 5 min and then coated with laminin solution (10 μg per ml) for 4 h. The substrates were placed in 24-well plates, and iPSC-NSCs were seeded into the wells at a cell density of 60,000 per well. After 6 days’ differentiation, the cells were fixed, and immunostaining on nuclei (DAPI) and neuronal marker (TuJ1) was conducted. Stem cell assays were repeated 3 times to obtain statistical information unless mentioned otherwise. Student t-test was used for two group analysis and ANOVA with Tukey post hoc test was used for multi-group (more than 3 groups) analysis. For the long-term (1 month, 30 days) stem cell differentiation assay, identical protocol was used with twice media change per week. Mature neuronal marker (MAP2) was used for identifying neurons differentiated from iSPC-NSCs on MnO 2 nanoscaffold.

Calcium imaging of neurons differentiated from iPSC-NSCs on MnO 2 laminin hybrid nanoscaffold in 12 well-plates. iPSC-NSCs were differentiated on MnO 2 laminin hybrid nanoscaffold using an identical protocol mentioned above for 6 days, then the cells were incubated with 1 ml of Fura-2 AM (Life Technologies, Catalog Number: F1201, 1:200 dilution, 5 μg per ml) in cell media for 1 h. Afterwards, cell media was changed to PBS. Under the movie mode of a fluorescence microscope, concentrated KCl solution in PBS (50 mM, 0.1 ml) was added to the cells, and the movie was taken for 10 min with 60 frames per seconds. The movies were pseudocolored, with red indicating strong calcium flux and green indicating weak calcium flux. An identical procedure was also applied for collecting calcium imaging of neurons differentiated from hNPCs. A summary of time dependent calcium intensity peaks can be found in Fig. 4, which was automatically obtained from the Nikon ND2 software.

Dye loading on MnO 2 nanoscaffold and MRI studies

To study drug loading and release on MnO 2 nanoscaffold, rhodamine B was used a model drug. Briefly, 0.3 mg rhodamine B (Alfa Aesar, Catalog Number: A13572) was added into 3.0 ml of MnO 2 nanosheet solution. After incubation at room temperature for 12 h, 5.0 ml PBS (PH = 7.4) was gradually added into the solution and RhB loaded MnO 2 nanosheet was centrifuged down at 3431 g for 5 min and extensively washed with PBS for 6 times to remove the residual RhB solution. Then the RhB-loaded MnO 2 nanosheet was re-suspended in 10 ml solution and re-assembled with laminin using the identical conditions for fabricating MnO 2 laminin hybrid nanoscaffolds. To monitor the dye hold-up, RhB-nanoscaffold was incubated with PBS for 12 h, then the fluorescence of the supernatant was detected by a fluorescence spectra (Varian Cary Eclipse). The dye loading was confirmed by degrading the RhB-nanoscaffold using 1.0 mg/ml ascorbic acid PBS solution. Instant appearance of pink color from RhB proves the loading of RhB inside nanoscaffold. RhB-nanoscaffold before and after degradation was also spotted in a glass slide in a close-proximity and then imaged in the fluorescent microscope. To test the correlation between MRI signals and RhB released, different amount of RhB-nanoscaffold [5, 2.5, 1, 0.5, 0.1 mg (from left to right in Fig. 5c)] were degraded with ascorbic acid (1.0 mg per ml) to form a homogeneous solution. Then the same solution in 96-well plates was used for MRI (Aspect’s M2TM Compact High-Performance MRI, 1T) measurement and fluorescence measurement under Nikon fluorescent microscope.

To study the day-dependent drug (RhB) release from our MnO 2 -laminin hybrid nanoscaffold, PBS with 10 μg per ml vitamin C was used to incubate the RhB loaded nanoscaffold, and was changed regularly every day. Fluorescence images (Nikon fluorescent microscope) were taken at Day1, Day2, and Day7, and the intensities from 3 different experiments were used to quantify the amount of RhB released. As a control, PCL polymer was dissolved with RhB and then formed a scaffold by drying at room temperature. Then the dye release was measured at the same time points as RhB loaded nanoscaffolds. The percentage of dye release was all normalized to the fluorescence intensity obtained at Day1.

To load neurogenic drugs into MnO 2 laminin hybrid nanoscaffold, DAPT (N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester, Tocris, Catalog Number: 2634) was first dissolved in a PBS: DMF = 9:1 solution (dimethylformamide, Sigma-Aldrich) at a concentration of 0.1 mg per ml. Then 1.0 ml of DAPT solution was quickly mixed with 100 μL of 3 mg per ml MnO 2 nanosheet aqueous solution. After incubating for 12 h, the solution was centrifuged down and washed with de-ionized water for 3 times. The successful loading of DAPT onto MnO 2 nanosheet was confirmed by MALDI-TOF (Bruker, Ultraflex) based on the Na+-DAPT peak at 455 (molecular weight to charge ratio). Briefly, 50 μL MnO 2 nanosheet loaded with DAPT solution was mixed with 50 μL gold nanoparticle solution (Ted Pella, 10 nm, 5.7 × 1012 particles per ml). Then 1 μL of the mixed solution was drop-cast onto ITO glass and baked at 50 °C for 1 min to fully evaporate water. The DAPT solution was drop-cast on the same ITO glass as a reference. ITO glass was placed into the MALDI-TOF and exposed with a laser for the analysis.

To measure the DAPT loading and releasing profile on DAPT MnO 2 nanoscaffold, 3.0 mg MnO 2 nanosheets were first loaded with DAPT using the previous protocol and then assembled into MnO 2 laminin hybrid nanoscaffolds. Using UV-Vis spectroscopy, we first identified the characteristic absorption peak of DAPT (20 μM in water with 1% DMF) at 264 nm. Then the drug loading amount was determined by the full disappearance of the 264 nm peak after incubated with nanosheets (2.0 ml PBS, 1.5 mg per ml). This corresponds to a loading efficiency of 110 μg per 3.0 mg nanosheets and a molar ratio between DAPT and manganese atom = 1:134 (Supplementary Fig. 13). After that, to quantify the release of DAPT, degradation and DAPT release from the nanoscaffold was initiated by incubating it in a solution containing 10 μg per ml ascorbic acid. As DAPT form strong binding complex with MnO 2 nanosheets (Binding Energy = −18.43 kcal per mol), we monitored DAPT release through quantifying the amount of manganese amount at different time points, and estimated the drug release based on the constant molar ratio between DAPT and manganese (1:134) in the MnO 2 -DAPT complex. The average daily release was quantified through dividing the total amount of manganese released by the length of degradation. The summarized DAPT release profile can be found in Supplementary Fig. 13.

DFT calculations on small molecule and MnO 2 binding

DFT calculations were carried out using the Quantum ESPRESSO software package. For the geometry optimization Perdew-Burke-Ernzerhof (PBE) functional along with D2 dispersion corrections were used59,60. The MnO 2 surface and the MnO 2 bound complexes were treated with DFT + U method. This is because conventional DFT functionals are unable to describe the strong correlation effect among the partially filled d states in Mn61. The Hubbard parameter ‘U’, is introduced for the Mn 3d electrons to describe the on-site Coulomb interaction, as given in the well-known GGA + U method62. The values of U = 4 eV and J = 0 eV for MnO 2 were adopted63. Spin-polarized calculations were performed since bulk MnO 2 has an antiferromagnetic ground state. The electron cores were defined using ultrasoft pseudopotential for all the elements and were extracted from the Quantum ESPRESSO main website (http://theossrv1.epfl.ch/Main/Pseudopotentials). For the k-point mesh, a γ-center was used. The wave function cutoff of 60 Ry and kinetic energy cutoff of 240 Ry were used in all the cases studied. The Gaussian smearing was turned so that the difference between the free energy and the total energy is less than 0.005 Ry per atom. The energy convergence was set to 1 × 10−6 a.u. and the force convergence threshold for the ionic minimization was set at 1 × 10−4 a.u.

The binding energies on the MnO 2 surface were calculated for a series of small molecules (Supplementary Table 1) and the DAPT drug molecule. The size of the cell was taken equivalent to the size of the MnO 2 surface that has 8 × 8 oxygen atoms at the periphery. The box size for the simulated system is 23 × 23 × 40 Å, and periodic boundary conditions are used. This condition was chosen to mimic the 2D MnO 2 surface. We first performed geometry optimizations for the bound complexes, with the resulting energy referred to as E complex . We then optimized the structures of isolated MnO 2 surface and the molecule of interest, obtaining their energies E MnO2 and E mol , respectively. The binding energy is defined as \(E_{\mathrm b} = E_ {\rm complex} - E_{\rm MnO_2} - E_{\rm mol}\). Negative E b indicates binding while positive E b indicates repulsion to the surface.

In vivo transplantation of hiPSC-NSCs

Spinal cord injury, transplantation of nanoscaffold and tissue harvest: All animal work was conducted following the regulation of the Institutional Animal Care and Use Committee (IACUC) at Rutgers University. The Notch1CR2-GFP transgenic mouse (Mus musculus) was used in this study. Adult mice that are 5–6 months old were picked for the spinal cord injury experiments. No difference was observed between male or female animals, and thus the gender was not specified. Animals are randomized without pre-knowledge of their behaviors and then assigned to different experimental groups without selection. Three mice were used for each group. Observers were blind to animal groups when performing experiments and analysis. During the surgery, initial anesthetization was performed with 5% isoflurane and then maintained at 2% Isoflurane. For hemisection, a laminectomy at T10~11 lateral section of spinal cord (length of gap ≈1 mm) was first performed. Then the dorsal blood vessel was burned with a cauterizer, and the spinal cord was cut from middle line toward the left using a #10 scalpel. Following induction of injury, bio-materials with iPSC-NSCs, laminin-coated and DAPT-treated PCL, or surgifoam was inserted into the wound site, and the muscle around surgical wound was sutured and skin is stapled using wound clips. A cell density of 1 million cells/cm2 was used for transplantation and mice were sacrificed 7-week after injury. For harvest, the spinal cords from the injured animals were obtained via microsurgical dissection. They were washed in 1× PBS and fixed with 4% (w/v) paraformaldehyde (PFA) for 24 h. Fixed tissues were washed again and then cryopreserved in 30% (w/v) sucrose for 48 h. Afterwards, the spinal cord tissue was embedded in cryo-preserving media (Tissue Tek® OCT compound) and kept frozen at −80 °C. Spinal cord sections were stained with PH3, GFAP, and F4/80 antibodies to analyze the long-term effect of nanoscaffold on SCI (Fig. 8). One-way ANOVA was used for multi-group analysis. Data represents mean ± s.d., n = 3, *P < 0.05, **P < 0.01 with Tukey’s post-hoc analysis. All tissue sections are in the center of the scaffold implants and then identified nearby the transplanted sites.

In vivo hiPSC-NSC-GFP transplantation assay

To study enhanced transplantation of iPSC-NSCs using our nanoscaffold, we further transplanted GFP labeled iPSC-NSC cells (iPSC-NSC-GFP) into a non GFP wild type C57BL/6 mouse strain64. To obtain GFP labeled cells, we transfected iPSC-NSCs with plasmid expressing EGFP using previously reported protocol (Supplementary Fig. 20). We confirmed the high transfection efficiency (>90%) and strong green fluorescence from iPSC-NSCs before seeded to the scaffold for in vivo cell transplantation using fluorescent microscope (Supplementary Fig. 20b). The surgical procedures for transplantation of iPSC-NSC-GFP into C57BL/6 were kept identical as the experiments on Notch1CR2-GFP mice and we repeated the immuno staining on tissue sections 1-week post transplantation and 1-month (30 days) post transplantation. In addition to the 3 groups [injury only (surgifoam insertion), 3D BHI nanoscaffold, and PCL cell group] evaluated in Notch1CR2 mice, we added 3 other important control groups to better support the therapeutic potential of our developed scaffold system: MnO 2 scaffold without laminin or DAPT but with cell transplantation (MnO 2 cell group), MnO 2 nanoscaffold with laminin and DAPT but without cell transplantation (MnO 2 DAPT group), and direct injection of GFP iPSC-NSCs with laminin (laminin cell group). All other conditions were kept identical to the experimental group (3D BHI nanoscaffold). Each group includes 3 animals (n = 3) to check reproducibility. We summarized the animal groups in Supplementary Fig. 20, and all the tissue analysis results were summarized in Fig. 7 and Supplementary Figs. 21–23. GFP, TuJ1 and MAP2 positive cells were first identified by automatic detection function in the NIS Nikon software (NIS element AR) then the amount of cells were recorded for making the graphs (Supplementary Methods). Percentage of Syn positive cells are quantified by first identify GFP + Syn + cells then divided by amount of GFP + cells in each section. Unpaired student t-test was used for two group significance analysis and one-way ANOVA was used for multi-group analysis. Data represents mean ± s.d., n = 3 unless described otherwise, *P < 0.05, **P < 0.01, ***P < 0.001. In Supplementary Fig. 20, when we count GFP + cells, we used the automatic detection function in the NIS Nikon software to identify GFP + cells shown in Supplementary Fig. 20e, then summarized the amount of GFP + cells at specific distance intervals (100 μm) in the sagittal sections. All tissue sections are in the center of the scaffold implants and then identified nearby the transplanted sites.

Data availability

The data that support the findings of this study are available from the authors on reasonable request. New data related to this manuscript can also be found on the following link: https://figshare.com/projects/A_Biodegradable_Hybrid_Inorganic_Nanoscaffold_for_Advanced_Stem_Cell_Therapy/29040.