Nanopatterned bioresorbable elastomeric scaffolds to promote neural,glial,and endothelial differentiation using human embryonic and induced pluripotent stem cells

Abstract

Bioresorbable nanopatterned scaffolds functionalized with polydopamine (PDA) and graphene oxide (GO) have been shown to promote the differentiation of murine neural stem cells (mNSCs) toward neural and glial lineages. Herein, we aim to evaluate the compatibility of these scaffolds for the culture and differentiation of both human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells. Our results indicate that PDA and GO scaffolds support the topographic alignment of hESCs and hiPSCs cultures, while preserving their pluripotency characteristics. Upon differentiation, PDA and GO scaffolds guide cell specification toward the neuroectoderm germ layer and the neural crest. This promotes enhanced differentiation into both neural and supportive glial cells of the central nervous system (CNS), as well as Schwann cells of the peripheral nervous system (PNS). Moreover, nanopatterned scaffolds also support the differentiation of hESCs and hiPSCs toward endothelial precursors. These findings establish a novel culture platform that enables combined differentiation pathways, potentially relevant for applications in personalized medicine and regenerative cell therapy.

Introduction

Traumatic injuries to the central nervous system (CNS) can be classified into those affecting either the brain or the spinal cord. Traumatic brain injuries (TBIs) are characterized by structural and physiological disruption of the brain integrity, ¹ representing the first cause of mortality and disability among young people, adults, and the elderly. Over 50–60 million individuals are affected every year, and current estimations predict that 50% of the world’s population will sustain a TBI during their lifetime.^2,3 Survivors of severe TBI often experience a significantly reduced life expectancy. ⁴ Spinal cord injury (SCI), on the other hand, is a prevalent condition affecting over 20 million people worldwide, with its incidence increasing by approximately 700,000 new cases each year. ⁵ SCI can be classified as traumatic or non-traumatic depending on the type of injury. Traumatic SCI can occur when the spinal cord is subjected to external physical impact, including traffic accidents (38%), falls (31%), gunshot wounds and other forms of violence (14%), sports and other recreational activities (9%), etc. ⁶ Non-traumatic, or primary, SCI typically begins with an initial mechanical insult that disrupts spinal cord physiology by impairing neuronal transmission. When the mechanical damage to the neuronal circuitry is severe, it leads to axonal alteration, progressive degeneration, and demyelination. These processes ultimately result in neuronal death and irreversible neurological dysfunction.

Current cell therapies offer a limited potential for functional restoration following CNS injury. This is primarily due to the absence of a supportive tissue environment, cell-type heterogeneity, and cell dispersion, hindering a functional recovery.^7,8 For all these reasons, neural tissue engineering represents a promising tool in regenerative medicine affecting the CNS and peripheral nervous system (PNS). This approach relies on the integration of biological elements with last-generation biomaterials that serve as scaffolds to guide and support cellular migration and/or differentiation, thereby promoting neurorehabilitation. In this context, smart biomaterials have shown great promise in neural tissue engineering, ⁹ as well as in promoting the neural differentiation of human stem cells from various sources, including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs).^{10 –12} In addition to the nervous system, vascular cells play a crucial role in ensuring a continuous supply of oxygen and nutrients to neural and glial cells. This support is essential for maintaining neuronal survival, proper tissue function, and overall homeostasis. ¹³ In fact, nervous and vascular components are structurally and functionally interdependent and together constitute the neurovascular system. Consequently, the recovery of the neurologic activity after injury requires the restoration of the neurovascular compartment as a whole. ¹⁴ Given their pluripotent identity, hESCs have been used in the development of therapies for neural tissue engineering aiming at treating various neurodegenerative disorders, such as Parkinson’s disease, Huntington’s disease and SCI. ¹⁵ Several of these approaches are currently undergoing clinical trials.^16,17 Compared to hESCs, hiPSCs offer key advantages, including greater availability and the elimination of certain ethical concerns associated with the use of hESCs. Moreover, several clinical trials involving hiPSCs have already been conducted, yielding promising results. ¹⁸ Besides, hiPSCs constitute a unique tool for personalized medicine in the screening and treatment of neurological pathologies, as they can be derived directly from the patient and differentiated into neuro-glial lineages. ¹⁹ For these reasons, the combination of hiPSCs and bioresorbable scaffolds is emerging as a promising therapeutic strategy for generating neural tissue replacement grafts aimed at treating neurological affections.^{20 –22}

Both natural and synthetic materials have been used to fabricate smart scaffolds that mimic the physiological and structural neural microenvironment. Giving the fact that the success of the implant relies on the appropriate cell-biomaterial interaction, precise control over the physicochemical, mechanical and surface properties of the scaffolds is essential to achieve the desired outcome. Ideally, bioresorbable scaffolds should be fabricated from FDA-approved compounds and meet the necessary standards for clinical applications, including biocompatibility, biodegradability, and suitability for manufacturing, storage and handling.^{23 –26} Some preclinical trials compared early programed dismantling bioresorbable poly(D,L-lactide) (PDLLA) bioresorbable scaffold versus metallic stent in a porcine coronary artery model. ²⁷ To this end, functionalized biodegradable polymeric scaffolds exhibit attractive features for promoting neurogenesis, neuroprotection, and the biological stimulation of neuronal, glial, and endothelial differentiation.^28,29 Among the various polymeric materials available, bioresorbable (co)polyesters derived from L-lactide and ε-caprolactone (PLCL) exhibit some intrinsic aspects that facilitate their biomedical application in neural tissue engineering and vascularization. Particularly noteworthy is the ability to taylor their degradative and mechanical properties, enabling the adjustment of these critical parameters to better match the neural environment.^{30 –33}

Morphological and topographical criteria also constitute relevant aspects that importantly exert an influence on neuro-glial and endothelial differentiation of stem cells.^34,35 Concerning this, nanopatterned surfaces have demonstrated their ability to regulate the biological behavior and function of hESCs, ³⁶ hiPSCs^{37 –39} and human neural stem cells (hNSCs),^{40 –42} controlling cell fate by guiding cell alignment, growth and migration direction, microcircuits development and neuro-glial differentiation. Moreover, recent studies have revealed that surface modification with polydopamine (PDA) and graphene oxide (GO) enhances the adhesion, growth, differentiation capacity and neurite outgrowth of hNSCs. Both compounds present inherent properties of hydrophilicity, stability, conductivity, flexibility, and biocompatibility, which may facilitate the neuro-glial differentiation of stem cells. Consequently, over the last years, PDA and GO compounds, either alone or in combination, have been investigated for the development of nerve conduit scaffolds aimed at enhancing conventional treatments against neural lesions.^{43 –48}

Based on these insights, our group previously developed two-dimensional bioresorbable nanopatterned scaffolds functionalized with PDA (NanoPDA) and GO (NanoGO) that promoted the oriented alignment, growth, migration and neuro-glial differentiation of murine neural stem cells (mNSCs) and progenitors. ⁴⁸ In the present work, we further explored the use of NanoPDA- and NanoGO-based nanopatterned biomaterials for the in vitro culture of hESCs and hiPSCs. These pluripotent stem cell sources possess the capacity to differentiate into both neural and vascular lineages. Our aim was to generate neural tissue grafts that closely mimic the in vivo structure and function of the neurovascular system, with potential applications in future clinical cell therapies.

Materials and methods

Fabrication of scaffolds

The commercially available biodegradable copolymer PURASORB PLC 7015 (Corbion, The Netherlands) (PLCL7015) composed of L-lactide and ε-caprolactone in a 70/30 molar ratio, with a weight average molecular weight (MW) of 154.6 kDa and dispersity index (DI) of 2.10, as determined by gel permeation chromatography (GPC), was used to fabricate the nanopatterned PLCL scaffolds. These scaffolds were coated with polydopamine (PDA) by incubation in a 2 mg/mL aqueous solution of dopamine-hydrocholoride (cat# H8502, Sigma-Aldrich, San Luis, MA, USA). Subsequently, graphene oxide (GO) was deposited onto the surface of the scaffolds by incubation in a 0.25 mg/mL aqueous solution (cat# 947–768-1, Graphenea, Spain) for 30 min, as previously described. ⁴⁸ Circular samples with a diameter of 6 mm were used for cell culture experiments.

hESC and hiPSC lines

hESC (ES-2) and hiPSC (KiPS-4F-1, CVCLC999) lines were obtained from the Spanish National Stem Cell Bank (Banco Nacional de Líneas Celulares, Instituto de Salud Carlos III). ES-2 cells were derived from one cryopreserved embryo (Caucasian, 46 XY) at day 6 of development (CMRB, Spain). KiPS-4F-1 cells were derived from epidermis keratinocytes (Caucasian, 46 XY) from 4 years-old individual (The Salk Institute for Biological Studies, USA). For cell passage and weekly maintenance, cells were mechanically disaggregated and picked up as small colonies using a cell stripper (Flexipet^® Adjustable Handle Set, Cook Medical, Bloomington, IN, USA), and cultured at an approximate concentration of 4–10 colonies/cm² on vitronectin-coated (cat# A31804, Life Technologies, Carlsbad, CA, USA) 6-well plates (cat# 3506, Corning, Corning, NY, USA) in Essential 8™ medium (E8, cat# A1517001, Life Technologies) at 37°C and 5% CO₂. To seed individual cells to conduct the experiments, cell lines were enzymatically disaggregated as single-cells using StemPro™ Accutase™ (cat# A1110501, Life Technologies) during 7 min at 37°C and 5% CO₂. For ectoderm differentiation, cells were disaggregated as for maintenance conditions (small colonies), and for neuro-glial differentiation, cells were disaggregated as for characterization assays (single-cells). Then, small colonies (4–10 colonies/cm²) or single-cells (1 × 10⁵ cells/cm²) were seeded by drop on vitronectin-coated coverslips (control), NanoPDA or NanoGO scaffolds placed on 24-well plates (cat# 3524, Corning) in E8 medium during 24, 48, and 72 h (characterization) or corresponding ectoderm or neuro-glial differentiation media. Cryopreservation was performed with 10% DMSO (#cat 20688, Life Technologies) in heat-inactivated FBS (cat# A5670701, Life Technologies) and a water bath set at 37°C was used for cell thawing. Cells were used at passages 10-20.

Prior to experimentation, karyotype analysis (G-band staining), HLA typing (NSG sequencing) and pluripotency and trilineage differentiation markers (RT-qPCR and immunofluorescence) were assessed. Besides, mycoplasma and sterility were evaluated monthly and annually, respectively.

Proliferation and viability assays

Cell proliferation was assessed with the CyQUANT™ XTT kit (cat# X12223, Life Technologies), following manufacturer’s instructions, and absorbance was measured in an Infinite^® M Nano+ microplate reader (Tecan, Männedorf, Switzerland) at 450 and 660 nm. Cell viability was analyzed with the Live/Dead Fixable kit (cat# L10120, Thermo Fisher Scientific, Waltham, MA, USA) in a FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA).

Reactive oxygen species (ROS) production

Intracellular ROS levels in cells cultured on nanopatterned scaffolds were assessed using the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate reagent (H₂DCFDA, cat# D399, Thermo Fisher Scientific). In brief, cells were incubated with 10 µM H₂DCFDA diluted in E8 medium during 30 min at 37°C and 5% CO₂. Then, cells were washed once with 1X PBS (cat# 20012-019, Life Technologies). Finally, fluorescence was measured in an Infinite^® M Nano microplate reader at 495 nm excitation and 527 nm emission.

Scanning electron microscopy (SEM)

Cells were fixed with 2% of glutaraldehyde (cat# 49629, Sigma-Aldrich) in Sorensen’s Phosphate Buffer (SPB, 0.1M, pH 7.4, cat# 100496, VWR International, Radnor, PA, USA) for 4 h at 4°C and washed 3 times with SPB for 30 min. Then, cells were dehydrated with ethanol and dried with hexamethyldisilazane (cat# 440191, Sigma-Aldrich). Finally, samples were coated with gold layer and visualized using a scanning electron microscope Hitachi S-3400 N (Hitachi, Tokyo, Japan).

Videomicroscopy

Prior to videomicroscopy, cells were seeded at a density of 1 × 10⁵ cells/cm² on NanoGO scaffolds in a petri µ-Dish 35 mm (Ibidi, Gräfelfing, Germany) and incubated for 30 min to allow cell attachment. Live-cell imaging was performed using a Nikon BioStation IM-Q videomicroscopy (Nikon Instruments Europe BV, Amsterdam, Netherlands) equipped with a 10× objective during 72 h. Imaging conditions were maintained at 37°C, 5% CO₂ and 95% relative humidity as previously described. ⁴⁸ Briefly, images of six fields per condition were taken every 15 min using a 10× objective. Tracking and overlay of individual cell tracks over a reduced period of 8 h were carried out using the Manual Tracking plugin in ImageJ software. The dynamic parameters (migration velocity, pausing time, persistence and total traveled distance) were calculated with a macro developed by Cordelières et al. (Bordeaux imaging center, UMS 3420 CNRS, Bordeaux, France). ⁴⁹

Differentiation assays

To differentiate hESCs and hiPSCs into ectoderm germ layer, STEMdiff Trilineage Differentiation Kit (cat# 05230, Stemcell Technologies, Vancouver, Canada) was used during 5 days to generate endoderm and mesoderm or 7 days to obtain ectoderm as previously described.^50,51

To differentiate hESCs and hiPSCs toward neural lineage, NeuroCult NS-A Proliferation and Differentiation Kits (cat# 05751 and cat# 05752 respectively, Stemcell Technologies) were used as previously described.^48,52 Briefly, cells were cultured with proliferation media supplemented with 1% Glutamax, 1% Penicillin/Streptomycin (P/S), 2% B27 without vitamin A (cat# 13462629, cat# 15140122 and cat# 12587-010 respectively, Thermo Fisher Scientific), 2 µg/ml heparin solution (cat# 07980, Stemcell Technologies), 20 ng/mL epidermal growth factor (EGF) and 10 ng/mL fibroblast growth factor-2 (FGF2) (cat# 130-093-825 and cat# 130-093-838, Miltenyi Biotec, Bergisch Gladbach, Germany) for 3 days. Then, culture medium was changed to differentiation media supplemented with 1% Glutamax, 1% P/S and 2% B27 with vitamin A (cat# 17504-044, Thermo Fisher Scientific) for 7 days. Fresh medium was replaced every 3 days.

RT-qPCR

Total RNA from cultured hESCs and hiPSCs was extracted and purified using RNAqueous™-Micro Kit (cat# AM1931, Invitrogen), and its concentration was determined by a Spectro-photometer (NanoDrop ND-1000). cDNA was synthetized utilizing Super-script™ VILO™ cDNA Synthesis Kit (cat# 10499763, Invitrogen). All samples were analyzed in triplicates, and each reaction included 9 µL master mix and 5 ng cDNA template with a final reaction volume of 10 µL. The master mix comprised gene-specific primers and iTaq Universal SYBR Green Supermix (cat# 1725271, BIO-RAD, Hercules, CA, USA). Pluripotency markers (OCT4, SSEA4, SOX2, KLF4, and NANOG), early ectoderm markers (OTX2, PAX2, BMP4, SOX1, and PAX6), neuro-glial progenitor markers (TUJ1 and NESTIN), immature neuronal marker (DCX), mature neural markers (NeuN, SYN1, and MAP2), glial markers (S100β, OLIG2, GFAP and Myelin Basic Protein: MBP) and endothelial related markers (CD31, CD34, KDR (VEGFR2), ID1, CDH5 (VE-cadherin), NOS3 (eNOS), VWF) were evaluated. Primers sequences and annealing temperatures are listed in Supplemental Table 1. RT-qPCR gene expression analysis was performed using the CFX96 Detection System (BIO-RAD) in the following conditions: a hot start at 95°C for 2 min followed by 40 cycles (denaturation at 95°C for 30 s, annealing for 1 min and elongation at 72°C for 30 s) and finalized by elongation at 72°C for 2 min. Data were analyzed utilizing the CFX Manager software according to 2^−ΔΔCt method, ⁵³ and values were normalized to the housekeeping gene (GAPDH).

Immunofluorescence and imaging

Cells were fixed with 4% of formaldehyde (cat# 28908, Life Technologies) for 20 min at room temperature (RT) and washed 3 times with 1X PBS for 5 min. Then, cells were permeabilized and blocked using 1X Tris-Buffered Saline (TBS, 50 mM Tris-Cl, pH 7.6; 150 mM NaCl) with 0.3% Triton X-100 (cat# 9002-93-1, Sigma-Aldrich) and 6% Donkey serum (cat# S30, Millipore, Burlington, MA, USA) for 30 min at RT. Primary antibodies were incubated for 1 h at RT (viability markers: Ki67 and CASP3) or overnight at 4°C (pluripotency markers: OCT4, SSEA4, SOX2, TRA-1-81, and NANOG; neuro-glial progenitor markers: TUJ1, GFAP, and NESTIN; immature neuronal marker: DCX; mature neural cell markers: NeuN, SYN1, and MAP2; glial cell markers: S100β, OLIG2, MBP, and P75^NTR; activation marker: c-FOS; endothelial cell marker: CD31). After three washes with 1X TBS, corresponding secondary antibodies were added for 2 h at RT. Antibodies used are listed in Supplemental Table 2. Actin fibers were stained with ActinRed™ (cat# R37112, Invitrogen) and the nuclei were counterstained with NucBlue™ (cat# R37605, Invitrogen). Images were acquired with a 20× objective of the Leica Microsystems DMi8 (Leica, Wetzlar, Germany) inverted microscope and counted in 10 fields per sample. Quantification of the length of neurite projections were done drawing the free-hand lines overlapping TUJ1 staining on the acquired images using the ImageJ software. Semi-quantitative analysis of fluorescence intensity for TUJ1, MAP2, MBP, and CD31 was performed using ImageJ software (NIH, USA) on random confluent fields. The mean gray value within each field was measured to determine the average fluorescence intensity. All images were acquired and analyzed under identical settings to ensure consistency across samples.

Statistical analysis

Statistical analysis was performed using GraphPad Prism statistical software (version 9.0). Data sets were first subjected to a Saphiro-Wilk test to verify that they fitted to normal distributions. Significance was assessed using the Student’s two-tailed unpaired t-test and ANOVA test, when appropriate. Data are expressed as the mean ± standard deviation (SD). All mean ± SD and p-values for RT-qPCR and fluorescence data analysis are included in Supplemental Table 3 and Supplemental Table 4, respectively. Each experiment was performed at least three times and a summary of the number of biological and technical repeats is included in Supplemental Table 5.

All data management were performed by following FAIR and CARE Data Principles.

Results

NanoPDA and NanoGO nanopatterned scaffolds do not interfere with the adhesion of hESC and hiPSC lines, allowing their growth in vitro

Human pluripotent stem cell cultures of hESC (ES-2) and hiPSC (KiPS-4F-1) were grown as colonies. Both cell lines were mechanically disaggregated and individualized using Accutase prior seeding onto the scaffolds. Cells were then seeded at a density of 1 × 10⁵ cells/cm² in E8 medium onto NanoPDA and NanoGO scaffolds to assess biomaterial interaction with the human stem cell cultures. To facilitate cell adhesion, NanoPDA and NanoGO scaffolds were pre-coated with vitronectin. A vitronectin-coated coverslip was used as a control. Then, cell number was determined during the first 72 h using XTT assay. Cell viability was analyzed using the Live/Dead assay, which demonstrated that disaggregated initial cultures started from a population of less than 50% of living cells. Cell number and viability was enhanced in the control with respect to NanoPDA and NanoGO scaffolds. The number of cells and their viability remained stable over the studied time points in both NanoPDA and NanoGO scaffolds, with no decrease in cell viability observed (Figure 1(a) and (b) and Supplemental Table 3). Endogenous ROS production has been previously associated to early differentiation state. ⁵⁴ Oxidative stress promotes exit from the stem cell state inducing spontaneous neuronal differentiation. ⁵⁵ To assess if the observed steady state could be the initiation of cell differentiation, ROS levels were measured with the cell-permeant DCFDA reagent. Our results showed that both ES-2 and KiPS-4F-1 human cell lines cultured on the nanopatterned scaffolds exhibited elevated ROS levels starting at 24 h, with a marked increase at 48 and 72 h (Figure 1(c)). Caspase activity has also been reported to mediate the differentiation of embryonic stem cells and, in some cases, to be dependent on the differentiation of neural stem cells.^56,57 Cleaved caspase-3 immunostaining was performed in ES-2 cells (Supplemental Figure 1A) and KiPS-4F-1 cells (Supplemental Figure 1B). Cleaved caspase-3 was selectively detected in the NanoGO condition in approximately 50% and 60% of ES-2 and KiPS-4F-1 cells, respectively, at 24, 48, and 72 h, despite no blebs or fragmented cell nuclei were observed (Supplemental Figure 1C–F and Supplemental Table 3). Cellular dynamics by videomicroscopy tracking was done in NanoGO conditions for ES-2 and KiPS-4F-1 cells. No significant differences were observed in their overall velocity, pausing time or distance traveled. However, differences in persistence time were detected, reflecting variations in migration behavior—specifically, in the average time between significant changes in the direction of cell translocation.^58,59 This variation could be explained by the different origin or source of cells (Figure 1(d)–(h) and Supplemental Table 3). Complementary to this, phalloidin staining revealed non-apoptotic morphology on cells cultured in NanoPDA and NanoGO versus control condition (Supplemental Figure 1G–H). Finally, since ROS are known to cause DNA damage, ⁶⁰ we expected an increase in Ki-67 protein levels, as Ki-67 protein synthesis is regulated not only during the cell cycle but also in response to DNA damage. ⁶¹ Over 80% of Ki-67 positive cells were found in our cultures, regardless of the cell type or culture conditions over time. ES-2 and KiPS-4F-1 cells seeded onto NanoPDA and NanoGO scaffolds expressed significantly lower Ki-67 immunostaining levels compared to control cells at 24 and 48 h, respectively, but no statistical significant differences were observed at 72 h (Supplemental Figure 1I’–J’ and Supplemental Table 3).

Figure 1.

Cell quantity does not increase over time for hESC and hiPSC cells grown with NanoPDA and NanoGO. (a) Estimation of total cell counts evaluated by XTT assay. (b) Cell viability analyzed by flow cytometry using the Life/Dead assay (c) ROS production detected by H₂DCFDA fluorimetry. Data are expressed as means ± SD. (d) Brightfield images shown the distribution of hESC and hiPSC of NanoGO videomicroscopy. Quantification of (e) mean overall velocity (f) percentage of the pausing time, (g) Total distance traveled and (h) Persistence time expressed in percentage for n = 60 cells per condition. (*) Comparison between NanoPDA/NanoGO and control conditions. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 10 μm.

NanoPDA and NanoGO nanopatterned scaffolds guide the alignment of hESC and hiPSC lines

Previous studies highlighted that substrate nanopatterning influences the differentiation capacity of human stem cells and, consequently, may impact on tissue regeneration.^48,62 Thus, we analyzed the orientation of ES-2 and KiPS-4F-1 cells individually seeded (1 × 10⁵ cells/cm²) on NanoPDA and NanoGO with E8 medium during 24 h. The actin cytoskeletal structures evidenced that both ES-2 and KiPS-4F-1 cells aligned with the topography of the nanopatterned scaffolds under both NanoPDA and NanoGO conditions. In contrast, control culture cells dispersed randomly (Figure 2(a) and (b)). This observation was confirmed by SEM micrographs, which indicated that both cell lines cultured on either NanoPDA or NanoGO scaffolds showed an elongated morphology aligned with the nanopatterned gratings, while under control conditions, both cell lines exhibited a more rounded cell shape. Furthermore, we also noticed that cells grown in NanoPDA or NanoGO presented a higher number of interacting cytoplasmic extensions compared to the control (Figure 2(c)).

Figure 2.

NanoPDA and NanoGO scaffolds induce the guided alignment of hESC and hiPSC lines seeded during 24 h. (a and b) ACTIN protein expression detected by ActinRed (phalloidin) staining. Scale bars, 100 µm. 2× insets zoom included in dashed boxes. Yellow arrows indicate cells trajectory over scaffolds. (c) SEM micrographs. Scale bars, 30 and 40 µm. 2× insets zoom included in black boxes.

NanoPDA and NanoGO nanopatterned scaffolds enhance the pluripotent capacity of hESC and hiPSC lines

Giving the essential role of pluripotency to define and support stem cell differentiation during tissue growth, remodeling and specialization, ⁶³ we determined the expression of several pluripotency specific protein and gene markers of ES-2 and KiPS-4F-1 cells seeded as single-cells (1 × 10⁵/cm²) on NanoPDA and NanoGO scaffolds with E8 medium during 24, 48, and 72 h. As shown in Supplemental Figures 2A–B, cells maintained their pluripotent identity in all culture conditions, as demonstrated by the conserved expression of OCT4, SSEA4, SOX2, TRA-1-81, and NANOG pluripotency proteins during time. Interestingly, NanoPDA and NanoGO scaffolds significantly enhanced the expression levels of OCT4, SSEA4, SOX2 and NANOG pluripotency genes in ES-2 cells compared to the control. However, we did not observe differences on KLF4 gene expression levels in ES-2 cells among the studied culture conditions. Similarly, KiPS-4F-1 cells seeded onto NanoPDA and NanoGO scaffolds expressed remarkably higher levels of SSEA4, SOX2, and NANOG genes compared to control cells. In addition, NanoGO KiPS-4F-1 cells showed a significantly higher expression level of KLF4 gene in NanoGO compared to the control, while OCT4 gene expression levels were similar between samples (Supplemental Figure 2C and Supplemental Table 3).

NanoPDA and NanoGO nanopatterned scaffolds select the differentiation of hESC and hiPSC lines toward ectoderm germ layer

Considering that pluripotent stem cells have the ability to differentiate into any cell type, we next determined the differentiation capacity of hESC and hiPSC lines into cells from the three germ layers. To achieve this aim, both human stem cell lines previously grown in E8 culture medium were mechanically disaggregated and seeded on NanoPDA and NanoGO scaffolds at 4–10 colonies/cm² using STEMdiff Trilineage differentiation media toward endodermal, mesodermal and ectodermal lineages. Culture medium was refreshed daily for up to 7 days (Figure 3(a)). Interestingly, we found that NanoPDA and NanoGO scaffolds induced the commitment of both cell types into early ectoderm, but did not promote the specification to endoderm and mesoderm layers. Thus, we analyzed the expression of distinct defined ectoderm markers at protein and genetic levels. For this purpose, cells were fixed and immunostained against neuronal (TUJ1) or astroglial (GFAP) markers characteristic of neuroectodermal specification. As observed in Figure 3(b) and Supplemental Figure 3A and B, both nanopatterned scaffolds supported the growth and spreading of both human cell lines, similarly to the control condition. Indeed, we noticed that both ES-2 and KiPS-4F-1 cells created three-dimensional (3D) multicellular structures over cell monolayer, exhibiting multiple long neural-like prolongations evidenced by TUJ1 staining (see yellow arrows Figure 3(b)). Contrarily, control cells comprised two-dimensional (2D) cultures that showed fewer neurite projections together with polarized structures forming neural rosettes (see “NR” Figure 3(b)). However, no statistically significant differences were found in neurite projection length (Supplemental Figure 3B–D and Supplemental Table 3). Hence, this data suggest that after differentiation, control cells remained in a more primitive neuroectoderm compared to NanoPDA and NanoGO conditions. At gene expression level, it is known that OTX2 is expressed in the brain ⁶⁴ and is a key regulator of the earliest stages of hESCs differentiation.^65,66 Our results showed that, although no significant changes were observed in ES-2 cell, KiPS-4F-1 cells showed an increase in OTX2 gene expression when cells were cultured on the nanopatterned scaffolds compared to the control, mainly in the presence of NanoGO (Supplemental Table 3). Next, we characterized the expression of the transcription factors involved in the development of the CNS (PAX2 and BMP4), neural progenitor cell formation and neurogenesis (SOX1), brain development (PAX6) and early stage neural (TUJ1) and glial (GFAP) differentiations. We observed an increase in gene expression associated with culture on nanopatterned scaffolds in both cell lines, indicating that the cells were differentiating into neural precursors and transitioning from a pluripotent to a neural progenitor state. Semiquantitative immunofluoresce intensity analysis also revealed consistent changes after 7 days (Supplemental Figure 10A and Supplemental Table 4). However, we observed differences depending on cell origin (Figure 3(c)). For example, ES-2 cells did not exhibit changes concerning PAX2 gene (0.02 ± 0.0002 (control), 0.02 ± 0.0005 (NanoPDA) and 0.02 ± 0.002 (NanoGO)) or GFAP gene expression (0.005 ± 0.0008 (control), 0.005 ± 0.0004 (NanoPDA) and 0.009 ± 0.003 (NanoGO)). Nevertheless, ES-2 cells showed an increase of BMP4 gene expression (3.13 ± 0.39 (NanoPDA) or 3.63 ± 0.32 (NanoGO) vs 1.74 ± 0.21 (control) (see Supplemental Table 3)); while showed a downregulation of PAX6 gene (1.36 ± 0.17 (control), 0.92 ± 0.11 (NanoPDA) and 0.83 ± 0.06 (NanoGO)), (control vs NanoPDA: p = 0.02201 and control vs NanoGO: p = 0.00754). These findings suggested a differentiation trend toward mesodermal and ectodermal lineages at the expense of neural specification. On the other hand, KiPS-4F-1 cells had no significant changes in BMP4 mRNA transcripts (3.47 ± 0.15 (control), 3.26 ± 0.13 (NanoPDA) and 3.30 ± 0.16 (NanoGO)) and a notable reduction of GFAP mRNA transcripts (0.003 ± 0.0004 (control), 0.001 ± 0.0002 (NanoPDA) and 0.001 ± 0.0002 (NanoGO)), (control vs NanoPDA: p = 0.00203 and control vs NanoGO: p = 0.00202). By contrast, PAX2 and PAX6 and SOX1 were upregulated in the NanoGO condition (Figure 3(c) and Supplemental Table 3). Interestingly the activation of these genes is known to be involved in ectodermal commitment in mouse ⁶⁷ and specifically in neural development at an early stage of neural differentiation. ⁶⁸

Figure 3.

NanoPDA and NanoGO scaffolds induce the ectodermal commitment of hESC and hiPSC lines. (a) Schematic representation of the protocol for trilineage differentiation. Image created with Biorender. (b) TUJ1 and GFAP proteins expression detected by immunofluorescence. Scale bar, 100 µm. Yellow arrows indicate neural-like prolongations. Dashed white lines comprise 3D cell aggregates. NR: neural rosette. (c) OTX2, PAX2, BMP4, SOX1, PAX6, TUJ1, and GFAP genes expression detected by RT-qPCR. Data are expressed as the means ± SD. (*) Comparison between NanoPDA/NanoGO and control conditions. (#) Comparison between NanoPDA and NanoGO conditions. *,^#p < 0.05, **,^##p < 0.01, ***,^###p < 0.001.

NanoPDA and NanoGO scaffolds accelerate the differentiation of hESC and hiPSC lines toward neuro-glial progenitors

For effective neural tissue repairing, newborn neurons and accessory glial cells are required. Accordingly, we tested the capability of ES-2 and KiPS-4F-1 cells seeded on NanoPDA and NanoGO scaffolds to differentiate toward neuro-glial progenitors. For this purpose, these human pluripotent stem cells were cultured in proliferation media for 3 days and then changed to neural differentiation media for 1 week. We analyzed the changes on cell phenotype at days 3, 7, and 10 (Figure 4(a)), focusing on both immunofluorescence staining of TUJ1 and NESTIN and the expression analysis of TUJ1, NESTIN, SOX1, and PAX6 genes that represent different stages in neural development. As exhibited in Figure 4(b) and (c) and Supplemental Figure 4A and B, double immunofluorescence of TUJ1 (i.e. marker of mature neurons, involved in the formation of their cytoskeleton) and NESTIN (i.e. expressed early during neural differentiation) showed a reduction of the NESTIN staining over time in both cell types. Furthermore, TUJ1 staining revealed that ES-2 cells cultured in control, NanoPDA and NanoGO conditions showed neural-like projections from day 7 of differentiation. However, albeit KiPS-4F-1 presented TUJ1+ bipolar cell morphologies, such structures became more evident at day 10 and only in NanoPDA and NanoGO conditions (see arrows of Figure 4(b) and (c)). Besides, as it occurred during the previously mentioned ectodermal specification, stem cells seeded on NanoPDA and NanoGO scaffolds originated more complex 3D cellular systems than control cells, which distributed in 2D monolayers. Next, we sought to determine changes in gene expression over time by RT-qPCR. TUJ1 was clearly upregulated in both cultures in NanoPDA and NanoGO scaffolds at day 10 compared to the control (Figure 4(d) and (e); Supplemental Table 3), which was further corroborated by semiquantitative mean fluorescence intensity analysis (Supplemental Figure 10B and Supplemental Table 4). Nestin staining represents the transition from pluripotent stem cells to neural progenitors. ES-2 cells seeded onto NanoPDA and NanoGO scaffolds downregulated NESTIN transcripts at day 3 and strongly increased at day 10, especially in the presence of GO (Figure 4(d) and Supplemental Table 3). Interestingly, in NanoPDA and NanoGO, NESTIN expression in KiPS-4F-1 cells showed a gradual increase at days 7 and 10 (Figure 4(e) and Supplemental Table 3). SOX1 is crucial for the formation of neural progenitor cells from pluripotent cells and helps to maintain the neural progenitor state, guiding the differentiation toward specific neural lineages. We observed a gradual increase of SOX1 in both cell lines cultured on NanoPDA and NanoGO scaffolds, which was accentuated by the presence of GO compared to the control at day 3, 7 and 10 of commitment (see Supplemental Table 3). Finally, we checked the evolution of PAX6 over the selected time-points, noticing a strong increase in both cell lines cultured in NanoPDA and NanoGO scaffolds at days 7 and 10 (Figure 4(d) and (e) and Supplemental Table 3). These data demonstrated that nanopatterned scaffolds were able to accelerate the differentiation of hESC and hiPSC lines toward neuro-glial progenitors.

Figure 4.

NanoPDA and NanoGO scaffolds accelerate the differentiation of hESC and hiPSC lines toward neuro-glial progenitors. (a) Schematic representation of the protocol for cell differentiation toward neuro-glial lineage. Image created with Biorender. (b and c) TUJ1 and NESTIN proteins expression detected by immunofluorescence. Scale bars, 100 µm. Yellow arrows indicate neural-like prolongations. (d and e) TUJ1, NESTIN, SOX1, and PAX6 genes expression detected by RT-qPCR. Data are expressed as the means ± SD. (*) Comparison between NanoPDA/NanoGO and control conditions. (#) Comparison between NanoPDA and NanoGO conditions. *,^#p < 0.05, **,^##p < 0.01, ***,^###p < 0.001.

NanoPDA and NanoGO nanopatterned scaffolds accelerate the differentiation of hESC and hiPSC lines toward neural stem cells and mature neurons

Our previous results highlighted that NanoGO scaffolds were able to accelerate neural differentiation of murine neural stem cells (mNSCs). ⁴⁸ Herein we sought to determine whether we could obtain similar results in human stem cells from different origins. Based on this, the protein expression of immature neuronal marker DCX and the mature neuronal nuclei marker NeuN was checked to assess the maturation progression in the course of neural specification. ES-2 cells cultured for 3 days on nanopatterned scaffolds showed a tendency toward enhanced NeuN immunoreactivity, along with DCX expression (Figure 5(a) and the same images with individual channels shown in Supplemental Figure 5(a)). However, monolayers of ES-2 and KiPS-4F-1 cells rapidly generated 3D multicellular aggregates at days 7 and 10 after growing on nanopatterned scaffolds, which became more evident for both cell types at day 10 (Figure 5(a) and (b) and the individual channels shown in Supplemental Figure 5A and B). Concerning DCX and NeuN mRNA expression, ES-2 cells showed a statistical significant upregulation of both genes on NanoPDA and NanoGO scaffolds compared to the control at days 7 and 10, mainly in PDA conditions (Figure 5(c) and Supplemental Table 3). Regarding the KiPS-4F1 cells, we detected an initial increase of DCX expression by cells in NanoPDA and NanoGO compared to the control at day 3 that was remarkably upregulated at day 10, especially in cells seeded on NanoPDA. In the case of NEUN, a notable upregulation was only observed at day 10 of differentiation, principally in NanoPDA conditions (Figure 5(d) and Supplemental Table 3). These data indicated that both cell lines exhibited the same tendency at day 10, strongly upregulating both immature and mature neuronal markers at the end of the neural differentiation process.

Figure 5.

NanoPDA and NanoGO scaffolds accelerate the differentiation of hESC and hiPSC lines toward neural stem cells and mature neurons. (a and b) NeuN and DCX proteins expression detected by immunofluorescence. Scale bars, 100 µm. (c and d) NeuN and DCX genes expression detected by RT-qPCR. (e and f) SYN1 and MAP2 proteins expression detected by immunofluorescence. Scale bars, 100 µm. (g and h) SYN1 and MAP2 genes expression detected by RT-qPCR. Data are expressed as the means ± SD. (*) Comparison between NanoPDA/NanoGO and control conditions. (#) Comparison between NanoPDA and NanoGO conditions. *,^#p < 0.05, **,^##p < 0.01, ***,^###p < 0.001.

The next step was to further characterize the neuronal differentiation of ES-2 and KiPS-4F-1 cells in greater detail. Thus, we analyzed the expression of microtubule associated protein 2 (MAP2) and Synapsin-I (SYN1). MAP2 stabilizes microtubules in neurons, contributing to the formation and maintenance of dendrites, considered essential for the proper functioning of neurons and synaptic integration. ⁶⁹ SYN1 is a presynaptic protein that regulates neurotransmitter release and plays a critical role in synapse formation and function. It is also involved in the synaptic vesicle cycle and contributes to synaptic plasticity. Immunostaining of SYN1 and MAP2 at days 3, 7, and 10 days in ES-2 cells was more prominent in 3D multicellular structures (Figure 5(e)). Interestingly, on both ES-2 and KiPS-4F-1 cells cultured on nanopatterned scaffolds, we observed again a faster generation of 3D multicellular structures positive for SYN1 and MAP2 proteins at days 7 and 10 of differentiation (Figure 5(e) and (f) and individual channels of the figure shown in Supplemental Figure 6A and B). RT-qPCR analysis in ES-2 cells showed significant increases on SYN1 mRNA transcripts at day 7, and very remarkably at day 10 (see Supplemental Table 3). Concerning MAP2 gene expression, RT-qPCR also highlighted a steady increase during neural cell differentiation for both NanoPDA and NanoGO (Figure 5(g) and Supplemental Table 3). On the other hand, for KiPS-4F-1 cells, RT-qPCR gene expression of SYN1 greatly increased in NanoGO compared to NanoPDA and control conditions at day 10 (Figure 5(g) and Supplemental Table 3) and MAP2 gene expression increased in both NanoPDA and NanoGO scaffolds compared to the control (Figure 5(h) and Supplemental Table 3). These findings suggest that NanoPDA and NanoGO nanopatterned scaffolds enhance the expression of neural differentiation markers in both hESC and hiPSC lines, with effects becoming evident after 10 days. Interestingly, both cell cultures grown on nanopatterned scaffolds exhibited higher fluorescence intensity compared to the control condition. This observation may be partially attributed to a slight background signal generated by the nanopatterned scaffolds themselves (Supplemental Figure 10B and Supplemental Table 4). Alternatively, despite the low mRNA expression levels, cells cultured on NanoPDA and NanoGO scaffolds may also activate regulatory mechanisms that enhance protein translation and/or stability. This effect could contribute to the increased fluorescence signal and is consistent with the enhanced neurite outgrowth observed.

Moreover, during differentiation from iPSCs, cellular specification is not fully synchronized. As a result, mRNA expression levels may remain unchanged across conditions, while some cells may have already initiated protein expression. This cellular heterogeneity could explain the observed differences in fluorescence intensity between the control and scaffold-based cultures.

NanoPDA and NanoGO nanopatterned scaffolds accelerate the differentiation of hESC and hiPSC lines toward glial cells from CNS and Schwann cells

Building on our previous finding that mNSCs cultured on nanopatterned scaffolds can give rise to cells from both neuronal and astroglial lineages, ⁴⁸ and after having demonstrated that both human ES-2 and KiPS-4S-1 cells are capable of commiting to a neuronal fate, we next investigated whether NanoPDA and NanoGO scaffolds could also support the growth of human glial cells. To this end, we checked by immunofluorescence the staining of mature astroglial marker S100β and oligodendroglial marker OLIG2 over time. As observed in Figure 6(a) and (b) (individual channels of the images shown in Supplemental Figure 7A and B), the presence of these proteins in both cell types was confirmed, mainly in NanoPDA and NanoGO conditions and at days 7 and 10 of differentiation, although to a lesser extent in KiPS-4F-1 cells. To fully corroborate the changes in glial cell specification associated to the presence of scaffolds, we performed RT-qPCR analysis of mRNA transcript expression that revealed a drastic upregulation of S100β and OLIG2 genes in ES-2 cells at day 7 (see Supplemental Table 3). Moreover, we noticed the same activation of such glial markers at day 10, especially in NanoGO conditions for both ES-2 and KiPS-4F-1 cells. These changes were accompanied by a progressive reduction in the gene expression of the immature astroglial and neural stem cell marker GFAP over the course of differentiation (Figure 6(c) and (d) and Supplemental Table 3).

Figure 6.

NanoPDA and NanoGO scaffolds accelerate the differentiation of hESC and hiPSC lines toward glial cells from CNS. (a and b) S100β and OLIG2 proteins expression detected by immunofluorescence. Scale bars, 100 µm. (c–f) S100β, OLIG2, GFAP, and MBP genes expression detected by RT-qPCR. Data are expressed as the means ± SD. (*) Comparison between NanoPDA/NanoGO and control conditions. (#) Comparison between NanoPDA and NanoGO conditions. *,^#p < 0.05, **,^##p < 0.01, ***,^###p < 0.001.

Furthermore, given the specific upregulation of OLIG2 gene expression at day 10 in both cell lines, particularly under the NanoGO condition, and considering the essential role of OLIG2 in early oligodendrocyte development and myelination, we next examined the expression of the myelination marker MBP. MBP is essential for the compaction and structural integrity of myelin. Its expression begins in oligodendrocyte precursor cells (OPCs) and increases progressively as these cells mature into fully differentiated oligodendrocytes. ⁷⁰ Analysis of MBP mRNA transcripts revealed a significant upregulation in cells cultured on NanoPDA scaffolds, and more prominently in ES-2 cells cultured on NanoGO scaffolds, compared to the control. This increase was particular evident at days 7 and day 10 (Figure 6(e) and (f) and Supplemental Table 3). In addition, such MBP gene activation induced by nanopatterned scaffolds was also detected in KiPS-4F-1 cells at day 10 (Supplemental Table 3, Supplemental Figure 10C and Supplemental Table 4). Interestingly, we again observed a shift in cell dynamics, with NanoGO emerging as the scaffold that primed lineage selection at day 7 for ES-2 and day 10 for KiPS-4F-1 cells (Figure 6(e) and (f), Supplemental Figure 10C, Supplemental Tables 3 and 4).

To corroborate these results, immunofluorescence of ES-2 on NanoPDA and NanoGO and KiPS-4F-1 cells on NanoGO at day 10 showed a marked positive staining for MBP protein (Figure 7(a) and (b), Supplemental Figure 10C and Supplemental Table 4) together with S100β and P75^NTR proteins, suggesting the differentiation into Schwann glial cells (see arrows of Figure 7(a) and (b)). In contrast, human stem cells maintained under control conditions did not exhibit such triple staining (data not shown), suggesting that both NanoPDA and NanoGO scaffolds support the generation of major glial cell types derived not only from CNS but also from PNS.

Figure 7.

NanoGO scaffolds accelerate the differentiation of hESC and hiPSC lines toward Schwann cells. (a and b) S100β, MBP and P75 proteins expression detected by immunofluorescence. Scale bars, 100 µm. Yellow arrows indicate Schwann cells.

NanoPDA and NanoGO scaffolds do not change Ki-67 cell expression but promote the differentiation to endothelial cells

Overall, both NanoPDA and NanoGO scaffolds supported heterogenous neural differentiation, yielding a mixed population of neuronal-like cells positive for Synapsin-I, DCX and MAP2, as well as Schwann glial-like cells expressing MBP, p75^NTR, and S100β. Previous studies have reported that sensory axons become functional at later stages in development, subsequently inhibiting Schwann cell proliferation through C-fos-mediated neuron-glial signaling. ⁷¹ Interestingly, C-fos has also been identified as a marker of neuronal activity, ⁷² and its overexpression has been associated with cell proliferation. ⁷³ Thus, we decided to analyze both cell proliferation and C-fos expression to unravel if our nanopatterned bioresorbable scaffolds could alter hESC and hiPSC cell behavior. Double immunofluorescence against Ki-67 and C-fos showed no differences at any time point analyzed (Figure 8(a) and (b) and individual channels shown in Supplemental Figure 8A and B). Moreover, Ki-67 staining in our differentiating culture media did not show significant temporal variation compared to the control (Figure 8(e)) and mild inconsistent changes of C-Fos were observed (Figure 8(f)). Altogether, all these results suggest that cells are still developing and are not yet fully mature and functional after 10 days in differentiating conditions.

Figure 8.

NanoPDA and NanoGO scaffolds promote the activation of hESC and hiPSC lines and stimulate their differentiation toward endothelial precursors. (a and b) Ki-67 and c-FOS proteins expression detected by immunofluorescence. Scale bars, 100 µm. (c and d) CD31 protein expression detected by immunofluorescence. Scale bars, 100 µm. (e and f) KI67 and c-FOS proteins quantification. (g) CD31 gene expression detected by RT-qPCR. Data are expressed as the means ± SD. (*) Comparison between NanoPDA/NanoGO and control conditions. (#) Comparison between NanoPDA and NanoGO conditions. *,^#p < 0.05, **,^##p < 0.01, ***,^###p < 0.001.

Previous literature has shown similarities between nervous and vascular development. ¹⁴ Besides, it has been suggested that PDA could create favorable microenvironments that promote endothelial differentiation.^74,75 Interestingly, it has also been reported that GO scaffolds could stimulate the differentiation and the proangiogenic activities of myogenic progenitor cells. ⁷⁶ Finally, it is also known that BMP4 protein, crucial for mesoderm induction, is often used to promote differentiation into various cell types including endothelial cells. ⁷⁷ For all these reasons, it could be plausible to have indirect cross-talk signals between these pathways that ultimately could balance the differentiation outcomes of pluripotent mesodermal stem cell cultures. This effect can be attributed to the presence of endothelial-promoting factors in the culture medium (e.g. VEGF and FGF2), which may bias differentiation toward the generation of CD31(PECAM-1)-positive endothelial cells. To check the presence of endothelial cells, we analyzed CD31 gene expression by RT-qPCR and immunofluorescence staining and fluorescent intensity against CD31 endothelial marker in both ES-2 and KiPS-4F-1 cells cultured at 3, 7, and 10 days either in control or NanoPDA or NanoGO scaffolds. We found some positive staining on ES-2 cultures (Figure 8(c)) and an increased staining on KiPS-4F-1 over time in the cultures on NanoPDA or NanoGO scaffolds (Figure 8(d), Supplemental Figure 10D and Supplemental Table 4). Individual channels of the images can be found in Supplemental Figure 9A and B. To fully characterize the onset of mRNA expression, we analyzed by RT-qPCR analysis the expression of CD31, observing a detectable significant upregulation at day 7 that becomes evident at day 10 for both cell types cultured on NanoPDA and especially NanoGO scaffolds (Figure 8(g). Statistics are detailed in Supplemental Table 3).

Previous works have shown that VEGF signaling through VEGFR2 is able to induce CD31 and VE-cadherin. ⁷⁸ VE-Cadherin is a key component of endothelial adherens junctions and together with the transcriptional regulator ID1 are involved in endothelial lineage commitment and differentiation. ⁷⁹ Indeed, blood-brain barrier-endothelial markers include CD31, VE-cadherin, and von Willebrand factor. ⁸⁰ To confirm the presence of endothelial cells and to try to clarify the mechanisms underlying lineage commitment and differentiation, we analyzed the expression of the endothelial progenitor marker CD34, the endothelial lineage commitment marker ID1 and the mature endothelial markers KDR (VEGFR2), CDH5 (VE-cadherin), and VWF (vWF) in both ES-2 and KiPS-4F-1 cells cultured at 3, 7, and 10 days either in control or NanoPDA or NanoGO scaffolds. In both cell types, the expression of KDR (VEGFR2), CD34, CDH5 (VE-cadherin), vWF, NOS3, and ID1 increased over time, with significantly higher levels observed in cells cultured on nanopatterned scaffolds compared to controls. Notably, NanoGO scaffolds consistently induced the highest expression levels across most markers (Supplemental Figure 11A and B and Supplemental Table 3). VE-cadherin, a key component of endothelial adherens junctions, showed a marked upregulation, indicating enhanced endothelial identity and cell-cell adhesion. ID1, a transcriptional regulator involved in endothelial lineage commitment, was significantly upregulated, particularly on NanoGO conditions, suggesting promotion of early endothelial specification. Despite our 2D system not allowing the characterization of functionality of capillary-like structures, we assessed NOS3 mRNA expression of eNOS, an endothelial cell-specific enzyme responsible for nitric oxide production, vascular tone regulation, and the regulation of angiogenesis.^81,82 We found NOS3 upregulated on NanoPDA and NanoGO scaffolds after 7 and 10 days in vitro, reflecting the potential functional maturation of endothelial cells. Finally, vWF, a marker of endothelial activation and hemostatic function, showed increased expression, supporting the acquisition of specialized endothelial traits (Supplemental Figure 11A and B and Supplemental Table 3). Altogether, these results suggest that nanopatterned scaffolds, especially NanoGO, enhance endothelial differentiation and maturation in both hES and hiPSC-derived cells.

Discussion

The ECM is increasingly recognized as an active regulator of stem cell behavior, acting not merely as structural support but as a dynamic and multivalent signal integrator that shapes cellular response. Previous studies have demonstrated that nanotopographical cues can influence stem cell fate through mechanotransduction pathways involving integrins, focal adhesion kinase (FAK), and downstream signaling such as MAPK/ERK and YAP/TAZ signaling pathways. This example of epigenetic regulation induced by the physical environment, may regulate neural and glial lineage commitment. ⁸³ Moreover, the physical signals received by cells can promote cytoskeletal organization and cellular polarity, which are critical during early neurogenesis. ⁸⁴ Scaffolds could facilitate induced cell polarization, which in turn could promote differentiation, since physical orientation guides the polarization of neural stem cells facilitating cytoskeletal organization and neurite outgrowth, which is crucial for cell differentiation. Accordingly, the use of polymeric scaffolds that can promote cell adhesion and cytoskeletal organization have attracted increasing interest to promote and/or accelerate the neural differentiation of hiPSCs and hESCs. For example, in the pioneering work by Lee et al., ⁸⁵ hESCs were differentiated toward the neuronal lineage in the absence of differentiation-inducing agents after 5 days thanks to the biophysical cues provided by the nanopatterned scaffold. ⁸⁵ However, no glial differentiation was observed, which may compromise the survival of neurons in the longer term. Inspired by this work, we herein developed similar supports based on generally-regarded-as-safe (GRAS) materials, that may facilitate a faster clinical translation in the future. Scaffolds fabricated via electrospinning, which generally require the use of potentially toxic organic solvents, have also demonstrated to be valid supports for the growth, differentiation, and integration of human hESCs and hiPSCs.^86,87 For example, as reported by Ghollasi and Poormoghadam, ⁸⁸ hiPSCs cultured onto laminin-functionalized polyethersulfone aligned electrospun scaffolds showed upregulated expression of neuronal and astroglial markers after 14 days in comparison to randomly aligned scaffolds. Despite the use of similar timepoints (i.e. 1 or 2 weeks) to assess neural differentiation in studies performed on 2D scaffolds, variations in culture protocols and conditions across these studies hinder direct comparisons. Moreover, recent studies have focused on developing 3D scaffolds that support the long-term culture, development, and maturation of hESCs and hiPSCs, with durations extending up to 120 days.^39,89

Our previous work based on the FDA-approved 2D bioresorbable nanopatterned scaffolds, functionalized with PDA and/or GO already showed cell polarization at short time after cell contact with the surface aligning the cells in the same orientation. ⁴⁸ In this study, we demonstrated that our bioresorbable nanopatterned scaffolds functionalized with PDA and GO are effective bioengineering platforms for promoting cell adhesion, guiding cell orientation and inducing the differentiation of human hESC (ES-2) and hiPSC (KiPS-4F-1) lines toward a neuroectoderm fate. At early time points, we found that the number of cells growing on the scaffolds did not increase significantly. However, the detected ROS levels in cells cultured on the scaffolds were significantly higher compared to those in control cells. Despite previous studies reporting that graphene and graphene oxide induce ROS production in human cells, ⁹⁰ we did not observe an additive or cumulative effect compared to NanoPDA alone. However, the production of ROS has been associated to early differentiation states, promoting the exit of the cell stemness.^54,55 Interestingly, we found that during the first 72 h, NanoGO increased the Caspase-3 expression in our stem cells. Previous works also reported the role of caspase activity to mediate the differentiation of embryonic and neural stem cells.^56,57 Our previous results also demonstrated that NanoGO scaffolds were able to accelerate the neural differentiation of murine neural stem cells. ⁴⁸

Despite the initial reduction in cell viability observed in ES-2 and KiPS-4F-1 cultures on nanopatterned scaffolds, likely due to the disaggregation and seeding steps, switching to differentiation medium led to a general increase in both TUJ1 gene and protein expression in scaffold-grown cells compared to control. Interestingly, BMP4 overexpression was also found in ES-2 cultures on both NanoPDA and NanoGO scaffolds with respect to the control. BMP4 upregulation has been previously reported to be necessary for the promotion of neural crest induction in human iPSCs. ⁹¹ Despite we did not found its increase in KiPS-4F-1 cells, we observed a strong SOX1 upregulation on both NanoPDA and NanoGO conditions. SOX1 has been previously reported to play a direct role in neural cell fate determination and differentiation ⁹² and its overexpression can induce neuronal lineage commitment. ⁹³ These divergences represent a difference in the pattern of intracellular signaling between embryonic and adult reprogramed stem cells to differentiate toward neural fate.

We also found that OTX2 and SOX1 had an increased expression in KiPS-4F-1 with respect to ES-2 cells when cultured in nanopatterned scaffolds, being their expression boosted in NanoGO condition. OTX2 belongs to a group of homeobox genes that are typically described as a head organizer in the primitive streak stage of embryonic development and has been involved in the regional patterning of the midbrain and forebrain, ⁶⁴ becoming a key regulator of the earliest stages of stem cell differentiation.^65,66 On the other hand, SOX1 is one of the earliest transcription factors expressed during the differentiation of hiPSCs into neuroectodermal lineages, indicating the onset of neural commitment. Both genes play a critical role in the specification and maintenance of neural progenitor cells (NPCs) and are often used as markers to confirm the successful differentiation of hiPSCs into neural progenitor cells. Indeed, the upregulation of SOX1 has been reported to occur concurrently with other neuroepithelial markers like PAX6 and NESTIN during hiPSCs differentiation to NPCs.^94,95 This is in agreement with our observed results of KiPS-4F-1 cells seeded on NanoGO scaffolds, thus confirming the boost of the induction of differentiation toward early neural progenitor status. Thus, our results suggest that hiPSCs are more suitable than hES for brain cell therapy and the use of nanopatterned scaffolds could reduce the amount of required compounds and complexity of the protocol, representing a time- and cost-effective approach. For example, differentiation protocol to generate basal forebrain-like cholinergic neurons from induced pluripotent stem cells (iPSCs) could last up to 53 days in vitro. ⁹⁵ Using nanopatterned scaffolds, we observed a marked upregulation of TUJ1, SOX1, and PAX6 genes expression levels within just 10 days in vitro.

Another advantage of using nanopatterned scaffolds is that the expression of gene markers corresponding to the various neural lineages was already detectable by 10 days in vitro: Neuronal (DCX, NEUN, SYN1 and MAP2) and glial (S100β, OLIG2 and MBP) markers were strongly upregulated in both ES-2 and KiPS-4F-1 human cells after 10 days in vitro with respect to the control. Interestingly, MBP is a key protein for myelin, primarily found in myelinating oligodendrocytes of the CNS ⁹⁶ and Schwann cells of the PNS. ⁹⁷ MBP is involved in the compaction and stabilization of the myelin sheath around axons, which is crucial for efficient nerve signal conduction.^70,98,99 Demyelinating diseases such as multiple sclerosis had an autoimmune loss of MBP. ⁹⁶ We found that our nanopatterned scaffolds strongly increased the MBP expression after 10 days in vitro with respect to the control. These results demonstrate that nanopatterned scaffolds are a valid alternative for human stem cell differentiation toward the main cell populations of the nervous system.

Nanopatterned scaffolds also supported the growth of Schwann cells expressing MBP, p75^NTR, and S100β, a coexpression pattern that has also been reported in the literature. ¹⁰⁰ S100β is a calcium-binding protein predominantly expressed in both non-myelinating and immature myelinating Schwann cells. Whether our cultures contain perysynaptic non-myelinating S100β⁺/NG2 (nerve/glial antigen 2) cells ¹⁰¹ remains an intriguing possibility that warrants further investigation. However, our differentiated cells showed an increased expression of the co-receptor p75^NTR, that together to S100β has been found to play key roles in neuroprotection, cell cycle regulation, differentiation, and response to injury.^102,103

Despite the elevated ROS levels observed during the initial days of culture on nanopatterned scaffolds, both ES-2 and KiPS-4F-1 cells reached confluence by 10 days in vitro without exhibiting signs of apoptosis (no blebs and no condensed nuclei). It has been previously reported that endothelial cells also participate in nerve regeneration promoting Schwann cells proliferation and migration, ¹⁰⁴ and the presence of vascular cells could improve demyelination. ¹⁰⁵ Interestingly, previous reports successfully differentiated endothelial cells from ES ¹⁰⁶ and hiPSCs. ¹⁰⁷ We therefore examined our cultures to determine whether the STEMdiff trilineage culture medium was permissive for the growth of CD31+ endothelial cells. Interestingly, we found an upregulation of CD31 mRNA expression at both 7 and 10 days in culture in cells cultured on nanopatterned scaffolds. It is well established that pluripotent stem cells transiently express markers from multiple lineages during early differentiation. This phenomenon is supported by evidence showing that paracrine signaling, particularly involving VEGF, VEGFR2, and VE-cadherin can induce endothelial fates even in non-mesodermal cells.^78,108 It is especially relevant the work of James et al., ⁷⁸ who demonstrated that VEGF, in combination with other factors, can directly promote the endothelial differentiation of human embryonic stem cells. The authors showed that VEGF signaling through VEGFR2 is essential for the induction of endothelial markers such as CD31 (PECAM1) and VE-cadherin, even in cells not yet committed to the mesodermal lineage.

Similar findings have been reported in early avian developmental stages, where VEGF secreted by neighboring cells can induce the expression of endothelial markers even in cells not originally committed to the mesodermal lineage. ¹⁰⁹ This underscores the critical role of paracrine signaling in directing cell fate decisions. Furthermore, during cardiomyogenesis, VEGF exposure has been shown to upregulate endothelial markers in cells derived from non-mesodermal layers, suggesting that this mechanism is conserved across differentiation pathways. ¹¹⁰ Taken together, these findings support the hypothesis that under specific microenvironmental conditions—such as those provided by surface functionalized scaffolds—VEGF signaling may be sufficient to trigger endothelial gene expression in ectodermal or otherwise non-mesodermal cells.

In addition to biochemical cues, mechanical and structural factors also play a role in regulating gene expression. The cytoskeleton, particularly actin dynamics, is a key regulator of cell morphology, polarity, migration, and transcriptional activity. Actin remodeling is known to mediate the transition between quiescent and angiogenic endothelial states, enabling features such as front-rear polarity, invasiveness, and enhanced motility—hallmarks of angiogenesis. ¹¹¹

Collectively, these mechanisms—paracrine signaling, cytoskeletal regulation, and scaffold-induced microenvironmental cues—may converge in our model to explain the observed endothelial-like phenotypes. These findings are consistent with the literature and suggest that the underlying regulatory pathways remain active and responsive in our experimental conditions.

Finally we checked the proto-oncogen C-Fos as it has been related necessary for both cell migration in Schwann cells ¹¹² and indirect transynaptic marker for neuronal activity. ⁷² However, no sustained differences between the nanopatterned scaffolds and the control were detected, suggesting that our scaffolds do not induce changes in basal neuronal stimulation.

In conclusion, our results strongly support the use of FDA-approved two-dimensional bioresorbable nanopatterned scaffolds as effective tools for promoting neural differentiation of human pluripotent stem cells, while also enhancing the production of Schwann and endothelial cells. These scaffolds provide structural support and offer a novel, fast, and easy alternative for in vitro cell culture. The resulting heterogeneous mixture of endothelial cells, neuronal cells, and glia may represent a promising cell cocktail for improving treatments of the nervous system injuries. Through implantation of the bioresorbable scaffold, this approach could become a versatile platform for cell therapy in the neural tissue repair. However, this application lies beyond the scope of the manuscript and will be investigated in future research.

Supplemental Material

sj-docx-1-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-docx-1-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-docx-2-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-docx-2-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-docx-3-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-docx-3-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-docx-4-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-docx-4-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-docx-5-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-docx-5-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-10-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-10-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-11-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-11-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-12-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-12-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-13-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-13-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-14-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-14-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-15-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-15-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-16-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-16-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-6-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-6-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

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sj-tif-7-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-7-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-8-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-8-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering

Supplemental Material

sj-tif-9-tej-10.1177_20417314251373038 – Supplemental material for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells

Supplemental material, sj-tif-9-tej-10.1177_20417314251373038 for Nanopatterned bioresorbable elastomeric scaffolds to promote neural, glial, and endothelial differentiation using human embryonic and induced pluripotent stem cells by Irene Romayor, Sara Martín-Colomo, Yurena Polo, Carlos Bello, Ruth Basanta-Torres, Irene Manero-Roig, Beatriz Pardo-Rodríguez, Jon Luzuriaga, Fernando Unda, Jose-Ramon Sarasua, Gaskon Ibarretxe, Aitor Larrañaga, Cristina Eguizabal and Jose Ramon Pineda in Journal of Tissue Engineering