Generation of induced pluripotent stem cells from rat fibroblasts and optimization of its differentiation into mature functional neurons

Abstract

Background:

Induced pluripotent stem cells (iPSCs) derived neural stem cells (NSCs) provide a potential for autologous neural transplantation therapy following neurological insults. Thus far, in preclinical studies the donor iPSCs-NSCs are mostly of human or mouse origin with concerns centering around graft rejection when applied to rat brain injury models. For better survival and integration of transplanted cells in the injured brain in rat models, use of rat-iPSC-NSCs and in combination with biomaterials is of advantageous. Herein, we report a detailed method in generating rat iPSCs with improved reprogramming efficiency and differentiation into neurons.

New Method:

Rat fibroblasts were reprogrammed into iPSCs with polybrene and EF1α-STEMCCA-LoxP lentivirus vector. Pluripotency characterization, differentiation into neuronal linage cells were assessed with RT-qPCR, Western blotting, immunostaining and patch-clamp methods. Cells were cultured in a custom-designed integrin array system as well as in a hydrogel-based 3D condition.

Results:

We describe a thorough method for the generation of rat-iPSC-NSCs, and identify integrin α_vβ₈ as a substrate for the optimal growth of rat-iPSC-NSCs. Furthermore, with hydrogel as the supporting biomaterial in the 3-D culture, when combined with integrin α_vβ₈ binding peptide, it forms a conducive environment for optimal growth and differentiation of iPSC-NSCs into mature neurons.

Comparison with existing methods:

Published studies about rat-iPSC-NSCs are rare. This study provides a detailed protocol for the generation of rat iPSC-NSCs and optimal growth conditions for neuronal differentiation. Our method is useable for studies to assess the utility of rat iPSC-NSCs for neural transplantation in rat brain injury models.

1. Introduction:

The dogma that the mature brain lacks regeneration capacity has changed since the discovery of neural stem cells (NSCs) in neurogenic regions in the brain, i.e., the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus, throughout the lifespan in mammals [1]. Apart from these neurogenic regions, NSCs can now be obtained by reprogramming somatic cells. The discovery of inducible pluripotent stem cells (iPSCs), which can provide large quantities of pluripotent cells with high plasticity capable of generating cells for all three germ layers including neurons and glia, has drastically changed the prospective of neural replacement therapy both in preclinical research and clinical practice [2, 3]. Compared to traditional embryonic derived stem cells, the somatic cell derived iPSCs are promising as they can be derived from patients themselves with potential for autologous transplantation, thus avoiding ethical and graft rejection concerns. Because of these advantages, the utility of iPSC derived NSCs for neural regeneration has been studied extensively [4–8].

In cell replacement therapy following brain insults, to achieve the goal of neural regeneration, transplanted NSCs need to be differentiated into functional neurons. Studies both in vivo and in vitro have found that neuronal differentiation of NSCs depends on the external cues provided and/ or present in the local microenvironment [9–11]. Following brain insults, scar tissue surrounding the injury site, which is composed of a glial cell produced dense extracellular matrix (ECM) network and inflammatory cells, has posed a major chemical and mechanical barrier for regeneration [12, 13]. Thus modulating the focal microenvironment where the iPSC-NSCs will be placed is critical for the survival and neuronal differentiation of the transplanted cells for neural regeneration.

In iPSC research, the majority of studies are focused on human or mice derived iPSCs [14–17]. In preclinical studies, rats are commonly used in modeling neurological injuries because of their relatively larger size for better surgical manipulation and their superior performance in behavioral studies, particularly for cognitive tasks [18, 19]. When human or mouse derived iPSCs are used as donor cells in rat neurological injury models, long term immunosuppressive treatment is needed to prevent graft rejection and this often interferes with the pathological progression of the injury model. Therefore, in rat neurological injury models, the use of rat iPSCs derived NSCs is necessary for the more accurate assessment of the potential of iPSCs as autologous cell source for neural transplantation.

Thus far, there are no commercially available rat iPSCs to assess the feasibility of autologous transplantation in rat brain injury models prompting us to generate our own rat iPSCs. Herein, we report our strategy in generating rat iPSCs by reprogramming the rat embryonic fibroblasts using lentiviral and adenoviral vectors. The generated iPSCs were characterized by immunostaining, western blotting, qPCR as well as in vivo teratoma studies. Neuronal differentiation, maturation and functionality of our rat iPSCs were validated by electrophysiological studies. Furthermore, as the local microenvironment plays a critical role in influencing the fate of transplanted cells, NSCs are often co-transplanted with biomaterials plus growth promoting factors to enhance their survival and function in the injured brain. In this study we also assessed the effect of integrin binding peptides and a hydrogel-based biomaterial in promoting the growth of iPSC-NSCs in 2D and 3D cultures.

2. Materials and Methods:

Animals:

All the experiments involving animals were approved by the Institutional Animals Care and Use Committee (IACUC), at Virginia Commonwealth University. Fisher 344 rats and severe combined immune deficient mice (SCID) were procured from the vendor (Charles River Laboratory, USA), and housed in our AAALAC approved animal facility, with a 12-hour light/dark cycle, water and food provided ad libitum.

2.1. Reprogramming rat fibroblast cells

The flow of study procedures was depicted in the Fig. 1.

Fig. 1.

Experimental flow chart

Rat fibroblasts from two sources were used to compare reprograming potential: 1) primary fibroblast isolated from ear and tail skins of adult Fisher 344 rats which were used for other studies; 2) cryopreserved rat embryonic fibroblasts (iX cells biotechnologies, San Diego, CA). To isolate fibroblasts from adult rats, pieces of skins from ears and tails were collected and cleaned with 75% ethanol followed by washing with PBS containing penicillin and streptomycin. The cleaned skins of ears/tails were further cut into small pieces and were allowed to attach to the bottom of a culture dish in DMEM containing 30% fetal bovine serum (FBS). After a week following plating, fibroblasts were harvested and confirmed by Vimentin (Dako, CA) expression with immunostaining.

For reprogramming, fibroblasts from both sources in their 2–3 passages were used. A total of 10⁵ cells were seeded onto the 0.1% gelatin coated 6-well plates in mouse embryonic fibroblast (MEF) expansion medium. After 24 hrs, polybrene and EF1α-STEMCCA-Lox P lentivirus (Millipore Sigma, St. Louis, MO) were added into the culture. The viral transfection was carried out using the recommended protocol from the manufacturer. The plate was incubated at 37°C for 24 hrs and transfected again with polybrene and the EF1α-STEMCCA-Lox P lentivirus vector. The infected cells were then trypsinized at the next day and plated onto monolayer feeder cells (Mytomycin c inactivated mouse embryonic fibroblasts). The complete stem cell medium (CSCM) was used for cell culture and changed every 48 hours. The CSCM was comprised of DMEM/F12 supplemented with N2 and B27 (Invitrogen, Carlsbad, CA), 1,000 U/ml of rat leukemia inhibitory factor (LIF, Millipore Sigma, CA), 3 uM of GSK3 inhibitor CHIR99021 (Sigma Aldrich, St Louis, MO), 1 uM of MEK inhibitor PD0325901 (Sigma Aldrich, St Louis, MO), knock out serum replacement (10%) and vitamin C (10μg/ml). After two weeks in culture, the colonies started to appear. Colonies were then manually picked and subcultured for 3 weeks.

The lentivirual transgenes integrated during the transduction process pose the risk of insertional mutagenesis and thus are not suitable for clinical application of iPSCs. Moreover, the expression of transgenes adversely affects the pluripotency and limits their differentiation potential [20]. To eliminate the exogenous transgenes from the lentivirual vector transfected iPSCs, the Adeno-Cre recombinase adenovirus (Vector Biolabs, PA) was used following the protocol in the STEMCCA Cre-Excisable Constitutive Polycistronic Lentivirus Reprogramming Kit (Millipore Sigma, CA) with minor modifications. Briefly, iPSCs were cultured in T25 flasks to 80% confluency, dissociated with 0.25% trypsin-EDTA, centrifuged and resuspended in CSCM. The calculated amount of adenoviral Cre recombinase achieving a multiplicity of infection (MOI) of 3000 was then added and incubated with cells for 6 hrs with shaking in every 2 hrs. After that, the cells were then seeded on top of the feeder cell layer (inactivated MEFs) in 10-cm culture plates. The medium was replaced the following day and every other day thereafter with fresh CSCM until colonies started to appear. Colonies were apparent after 2 weeks and the individual colonies were picked up manually.

2.2. Expansion and characterization of rat-iPSCs

The established rat-iPSCs (riPSCs) were propagated on mitomycin-c inactivated mouse embryonic fibroblasts (MEFs) using CSCM and trypsinized (0.05% trypsin-EDTA: Sigma Aldrich, St. Louis, MO) and plated into new wells with MEFs feeder cells every 3 or 4 days. The characterization of generated rat iPSCs was determined by the following tests:

1. Alkaline Phosphatase expression test: The tests were carried out following the manufacture’s protocol using the alkaline phosphatase detection kit (Milipore Sigma, MA). Briefly, the colonies of iPSCs after 5 days culture were first fixed with 4% paraformaldehyde (PFA) for 2 minutes, then rinsed with Tris buffered saline followed by incubation with the alkaline phosphatase reagent for 15 minutes in the dark. After that, the pink stained alkaline phosphatase positive colonies were counted under microscope.

2. Quantitative real-time PCR analysis: The characterization of generated riPSCs was determined by gene expression analysis of pluripotent genes such as SOX2, Nanog and KLF4. The total RNA was isolated using Rneasy mini kit (Qiagen, MD), and cDNA was synthesized from 2ug of total RNA using iScript cDNA synthese kit (Bio-Rad, CA) following the manufacturer’s protocol. The Amplification of cDNA of riPSCs and rat embryonic fibroblasts was performed using iTaq Universal SYBR Green supermix (Bio-Rad, CA) in triplicate. The CFX 384 touch real-time PCR detection system and CFX Maestro software (Bio-Rad, CA) was used for the gene expression. The gene expression level of Nanog, SOX2 and KLF4 was assessed using the validated primers of these genes (Qiagen, MD) and β-actin, the house keeping gene.

3. Western blotting analysis: Protein expression level of iPSC related proteins was assessed using Western blotting. Briefly, the cell lysates from the rat iPSC and rat embryonic fibroblasts (REF) were prepared in RIPA buffer. The total protein level from these cell lysates was estimated using the BCA assay. For Western blotting, 20μg of protein was loaded into each well of a 4–12% SDS-PAGE Criterion Gel TGX stain-free gel (Bio-Rad, CA). The gel was activated using the Chemidoc MP imaging system (Bio-Rad, CA) for 45 seconds before blotting. The transfer of protein was performed on a PVDF membrane using the Trans-Turbo Blot transfer system (Bio-Rad, USA). The following primary antibodies were used: Anti-SOX2 (Bioss, CA), Anti-Oct4 (Novus Biologicals, CO), Anti-Nanog (EMD, Millipore, CA). The western blotting images were analyzed using image lab 6.0 software (Bio-Rad, CA). The stain-free images of the blot were used for normalization with chemiluminescent images.

4. In vivo Teratoma formation assessment: For this study, 3 male SCID mice at 8-week old were used. Briefly, one million rat iPSCs were mixed with a cold solution of matrigel/PBS (1:1 ratio), the mixture was then subcutaneously injected into the thigh region of the hind-leg of the mice. The mice developed masses at the injection site with time, and were perfused with PBS and 4% PFA at 4 weeks after cell injection. The subcutaneous tumor mass was dissected and fixed in 4% PFA. The tissues were paraffin embedded, sectioned and stained with Hematoxlin & Eosin (H&E). The histological assessment was conducted using an Olympus Microscope.

2.3. Differentiation of riPSCs into neural stem cells (iPSC-NSCs) and mature neurons

Differentiation of riPSCs into neural stem cells was performed following a published protocol with minor modifications [21]. Briefly, when the iPSCs were grown to 80% confluency, the CSCM was replaced with the neuronal induction medium containing Neurobasal-A (Life Technologies, Carlsbad) supplemented with 1X B27-A and 1X N2 (Life Technologies, Carlsbad, CA), 1XNEAA (Life Technologies, Carlsbad), 1X glutamax (Life Technologies, Carlsbad, CA), and 5 mg/mL of penicillin/ streptomycin. Every 3 days half of the media was replaced with freshly made media. Neurosphere formation was observed around 2 weeks of culturing. The detached neurospheres were then centrifuged and dissociated with the Accutase (Sigma Aldriach, St Louis, MO) and suspended in neural stem cell medium consisting of Neurobasal-A supplemented with 1X B27-A, 1X N2 (Life technologies, Carlsbad, CA), 1X NEAA, 1X Glutamax (Life Technologies, Carlsbad, CA), 20 ng/mL epidermal growth factor (EGF; Gibco, CA), 10 ng/mL basic fibroblast growth factor (bFGF; Gibco, CA), and 5 mg/mL penicillin/streptomycin (Gibco, CA). The neurospheres were further passaged every week for 3 weeks to convert into neural stem cells. The neural stem cells were confirmed via immunostaining with markers for neural lineage including Nestin (1:500; Millipore Sigma, CA), synaptophysin (1:1000, Abcam, MA), NeuN (1:500, EMD Millipore, CA), TUJ1 (1:1000, Biolegend, CA), MAP2 (1:1000, Millipore Sigma, CA), GFAP (1:1000, DAKO, USA) and Vimentin (1:500, DAKO, USA).

To further differentiate neural stem cells into mature neurons, the neural stem cell media consisting of neurobasal medium supplemented with B-27 and Glutamax-1 was used following the manufacture’s protocol (Thermo-Fisher Scientific, Waltham, MA) with some modifications. In order to accelerate the differentiation process, dibutyryl cAMP (Sigma Aldrich, St Louis, MO) was added (0.5 mM). The cells were continuously grown on poly-L-ornithine coated coverslips for 30 days with the medium changed every 3 days. Cells were fixed and stained for neuronal markers including TUJ1, MAP2, Synaptophysin and NeuN.

2.4. Electrophysiological recording

Parallel cultures of NSC neuronal differentiation were used for cell functional characterization with patch clamp recording after 30 days plating as described above. Briefly, the coverslips with cells were placed on the stage of an Olympus BX51WI microscope (Olympus, Waltham, MA). Cultures were continuously perfused in a chamber solution with saline consisting of (in mM): NaCl 125, KCl 3.0, CaCl 1.2, MgCl₂ 1.2, NaHPO₄ 1.2, NaHCO₃ 25, glucose 25 bubbled with 95% O₂ / 5% CO₂ and maintained at 32–35°C. Cell were then visualized with a 20x (0.95 N.A.) objective, images captured with a DAGE-MTI IR1000 CCD camera (Michigan City, IN), and displayed on a Panasonic high resolution black and white monitor for targeted recordings. Whole cell patch clamp recordings from iPSCs derived neurons were performed using patch pipettes pulled from borosilicate glass (8250 1.65/1.0 mm, King Precision Glass, Claremont, CA) on a Sutter P1000 pipette puller (Sutter Instruments, Novato, CA) filled with (in mM): KMeSO₄ 140, NaCl 8, MgATP 2, NaGTP 0.1, HEPES 10. Membrane potential responses to current injections (600 ms duration, −50 to +100 pA in 10 pA steps) were measured with a Model 2400 patch clamp amplifier (A-M Systems, Port Angeles, WA) and converted into a digital signal by a PCI-6040E A/D board (National Instruments, Austin, TX). WCP Strathclyde Software was used to store and analyze membrane potentials on a PC computer (courtesy of Dr. J Dempster, Strathclyde University, Glasgow, Scotland). Calculated junction potentials (9.4 mV) were compensated for in the analysis. Further analysis was performed with the Julia programming environment, OriginPro 2018 (OriginLab Corp., Northampton, MA, USA) and Graphpad Prism (San Diego, CA). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

2.5. Growth of rat iPSC-NSCs in integrin array plate and in hydrogel based 3-dimensional culture condition

To assess the influence of the environment on iPSC-NSC neuronal differentiation and maturation, we tested the growth of cells in 2D culture using a custom-designed integrin-binding array platform containing short synthetic peptides that bind to 16 types of integrins commonly expressed on almost all cells in vertebrates as well as in hydrogel-based 3D culture following our established protocol [22]. For 2D culture, 5,000 cells / well were seeded into a 48-well integrin-binding array platform with neural stem cell differentiation medium for 30 days as described above. For 3D culture, the integrin binding peptide that showed best support for the growth of iPSC-NSCs on the 2D integrin-binding array screening test was immobilized in a polyethylene glycol (PEG)-based injectable hydrogel. The experiments were performed in triplicate in a 48-well plate pre-coated with 20μl of hydrogel and incubated at 37 °C for 30 minutes before seeding cells (5,000 cells/well). The custom-designed 0.8% hydrogel was diluted to 50% using the selected integrin-binding peptides with iPSC-NSCs in the neural stem cell differentiation medium. The peptide stock solution was made in 1X PBS as 4 mg/ml and the final concentration in the culture was adjusted to 0.5 mg/ml. The components of culture included: iPSC-NSCs, selected integrin peptide, medium and hydrogel. All components were mixed as a cocktail thoroughly before adding into the wells of the hydrogel pre-coated 48-well plate. 50 μl of prepared cocktail was added into each well, followed by incubation at 37 °C for 30 minutes. Once the gel was fully set, 200 μl of differentiation medium was gently added from the top into the wells. The culture was maintained for 30 days with half of the media changed every 48 hours. After 30 days, the cells were fixed with 4% PFA for 3 hours and processed for immunostaining for TUJ1, MAP2 and DAPI. Images were captured using an LSM 710 Confocal Microscope (Carl Zeiss Microscopy, Germany).

2.6. Immuno-fluorescence staining and cell quantification

The immunostaining for different cell markers was performed following our established protocol [22]. Briefly, 4% PFA fixed cells were blocked with 10% horse serum for 1 hour followed by incubation with the primary antibodies described above overnight at 4 °C with agitation. Following washing with PBS, the cells were incubated with the secondary antibodies: Alexa Fluor 488 anti-rabbit IgG or Alex Fluor 568 anti-mouse IgG (both 1:200; Invitrogen, Waltham, MA). After incubation for 1 hr at room temperature, cells were then washed with PBS 3 times 10 minutes each then incubated with DAPI (1:1000) for 10 minutes before final washing and cover slipped with Vectashield.

For quantification of the percentage of TUJ-1 positive cells, 5 randomly selected fields per well were viewed and captured with a 20X objective using an inverted Olympus fluorescence microscope. The percentage of Tuj-1 positive cells was quantified as relative fluorescence intensity using Image-J software (NIH). Neurite outgrowth of TUJ-1 positive cells was traced according to our published protocol [22].

2.7. Statistical Analysis:

For data analysis of two-group comparison, the unpaired student t-test was used. For multiple group comparison, one- way ANOVA was used followed by post hoc test with Tukey’s test or Bonferroni correction. GraphPad Prism, 7.0 software was used and data were presented as mean ± SEM in all figures. Statistical significance was considered with p<0.05.

3. Results

3.1. Derivation of iPSCs from rat fibroblasts

The single polycistronic lentivirus vector system that we used contains transcription factors of Oct4, Klf4, SOX2 and C-Myc (OKSM) which not only reduces the number of viral integrations, but also significantly increases the downstream differentiation potential of iPSCs in vitro [23]. In this study, this vector system was effective in reprogramming of rat fibroblasts into iPSCs following two phases of viral transduction. The first viral transduction (Lentivirus) delivered the OKSM to the fibroblasts, followed by a second round of Adenovirus containing Cre to remove the viral transgenes from the generated iPSCs. Using this viral vector kit, we tested the efficacy of reprogramming both adult derived fibroblasts and embryonic fibroblasts. A robust reprogramming efficiency, as assessed by expression of alkaline phosphatase, was found with embryonic fibroblasts (Fig. 2), whereas the efficiency was much lower with the adult fibroblasts (data not shown). Due to the low efficiency of reprogramming with fibroblasts derived from the skin of adult rats, subsequent generation and characterization of iPSCs were performed using the colonies derived from the rat embryonic fibroblasts.

Fig. 2. Generation of iPSCs from rat fibroblasts.

a). Rat fibroblasts were identified with Vimentin staining. b-e). The rat embryonic fibroblasts were reprogrammed into iPSCs using a lentiviral vector containing four Yamanaka factors (OKSM). Generated iPSCs were then modified and lentiviral transgenes were removed using an adenovirus vector. The stem cells-like colonies appeared in two weeks (b: lower magnification; c: higher magnification). The iPSCs were confirmed by expression of alkaline phosphatase (AP). Approximately 90% of the colonies were AP positive (d: lower magnification; e: higher magnification). Scale bar =100μm.

To confirm successful reprogramming of rat fibroblasts into iPSCs, we assessed several typical iPSCs markers at protein and gene expression levels in our generated colonies using immunostaining, Western blot, and RT-qPCR. Immunostaining of the colonies revealed strong expression of both Sox2 and Oct4 (Fig. 3 a–b). In Western blotting, a significantly higher expression level of Sox2, Oct4 and NANOG was found in protein lysates from iPSC colonies in comparison to that from rat embryonic fibroblasts (REF) (Fig. 3 d). RT-qPCR revealed a significantly higher level of gene expression of Sox2, NANOG, and KLF4 in samples from iPSC colonies (Fig. 3c). These results have confirmed the pluripotency of the iPSC colonies generated from the rat fibroblasts.

Fig. 3. Characterization of rat fibroblasts derived iPSCs.

a-b). The iPSCs were cultured, fixed and stained for Oct 4 and Sox2 (red), DAPI, nuclear staining stained blue (a: on the top of the monolayer of feeder cells as colonies; b: on the matrigel coated plate; bar=100μm). c). RT-qPCR assessing gene expression levels of SOX2, NANOG and KLF4 in iPSCs in comparison to rat embryonic fibroblasts with fold expression changes between the two. d). Western blotting assessing protein expression level of pluripotency associated proteins of Sox2 (32KD), Oct4 (40KD) and Nanog (39KD) in iPSCs in comparison to rat embryonic fibroblasts (REF). The expression level of Sox2, Oct4 and Nanog was significantly higher in the iPSCs than the REF. e). A panel of representative images showing teratoma formation of rat iPSCs in an SCID mouse. H&E stained teratoma sections demonstrated tissues from three germinal layers of a typical embryo such as the ectoderm derived glandular tissues and duct system, the mesoderm derived striated muscle tissue and the endoderm derived simple ciliated columnar epithelium typically observed in the gastrointestinal tract. Scale bar =100μm.

Additionally, we also verified the pluripotency of the REF-derived iPSC colonies using in vivo teretoma formation assay in SCID mice. In all three mice which received iPSC injection, a subcutaneous tumor was formed at the site of cell injection. Histological examination with H&E staining showed that the tumor contained various tissues from all three germinal layers (Fig. 3 e), thus further confirming the pluripotency of the reprogrammed cells.

3.2. Differentiation of rat iPSCs into neural stem cells and mature functional neurons

The iPSCs are pluripotent cells capable of differentiation into cells of all three germinal layers. For iPSCs differentiation into neural stem cells (NSCs), iPSCs were grown in neural induction medium containing bFGF and EGF. Neurospheres were formed from iPSCs after two weeks of culturing in the neural induction medium (Fig. 4a). When neuropheres were processed for immunofluorescence staining with NSC markers Nestin, Sox2 and GFAP, the majority of cells in the neurospheres expressed these NSC markers confirming successful generation of NSCs from the iPSCs (Fig. 4b and c).

Fig. 4. Differentiation of iPSCs into neural stem cells.

a). Phase contrast images showing neurospheres at varying sizes. b and c). Immunofluorescence staining with NSC markers of Nestin (green), SOX2 and GFAP (red) and DAPI as a nuclear staining (blue). Scale bar =100μm.

To further differentiate the rat iPSC-NSCs into mature neurons, neurospheres were plated on the poly-L-orinithine (PLO) and laminin coated coverslips. After 5 weeks in culture, the majority of NSCs differentiated into mature neurons as demonstrated by expression of neuronal markers including presynaptic protein synaptophysin and neuronal nuclear antigen NeuN (Fig. 5a). To confirm the functionality of these cells, we patch-clamped 28 cells in a parallel culture of the cells that had been cultured for 5 weeks. Of the 28 cells recorded, 6 cells generated action potentials in response to depolarizing current injection. Of the 6 electrically excitable cells, 4 were stable enough to measure the electrophysiological properties of each cell. Only 1 cell generated trains of adapting action potentials (Fig. 5b) whereas the others generated just 1 action potential per current pulse (Fig. 5c). We next examined the effect of the voltage-dependent sodium channel blocker tetrodotoxin (1μM, TTX) in 2 of these cells. In each cell, TTX blocked the generation of action potentials. Injection of larger currents could not recruit an action potential (Fig. 5d). On average, action potentials had long durations (4.1 ± 0.4 ms), short amplitudes (43.3 ± 4.3 mV), and relatively depolarized thresholds to activation (−35.8 ± 1.3 mV). The passive properties of the cells had average resting membrane potentials of −58.7 ± 7.9 mV (range −49.4 to −74.4 mV), high input resistances (787.4 ± 226.1 MΩ), time constants of 23.3 ± 4.6 ms, and membrane capacitance measures of 33.8 ± 13.7 pF. The presence of a depolarizing sag in the membrane potential resulting from hyperpolarizing current injections (indicative to the presence of an Ih current) was variable. However, on average, cells had sag ratios (membrane potential amplitude at the end of the current pulse / peak membrane potential response) of 0.86 ± 0.8 (range 0.63 to 0.97). Given the variability of the electrically excitable cells’ electrophysiological properties, our data suggest that these cells were of different phenotypes or at different stages of development. Nevertheless, this data demonstrates that our rat iPSCs-NPCs are capable of differentiating into mature functional neurons.

Fig. 5. Maturation of rat iPSCs-NSCs into mature neurons.

a). Representative confocal images of differentiated neurons from rat iPSCs-NSCs grown on poly-l- ornithine and laminin-coated cover slides for 5 weeks expressing mature neuronal markers synaptophysin (red), NeuN (green) and DAPI (blue). Scale bar =20μm. b-d). Single cell patch clamp recording demonstrated the functionality of differentiated cells at 5 weeks with a subpopulation of iPSCs derived neurons demonstrated electrical excitability: b). An example of a neuron that produced a depolarizing sag in response to hyperpolarizing current injection (−40 pA) and a train of action potentials in response to depolarizing current injection (+60 pA) (b); c). Line plot illustrating the instantaneous frequency of action potentials resulting from different magnitudes of depolarizing current injection, and for all current amplitudes, the instantaneous frequency of action potentials displayed adaptation; d). An example of another iPSC derived neuron that produced an action potential to depolarizing current injection (left, 30 pA), and application of tetrodotoxin (1 μM, TTX) blocked action potential generation.

3.3. The α_vβ₈ integrin binding peptide promoted growth and neuronal differentiation of rat iPSC-NSCs in hydrogel-based 3D culture system

As environment is crucial for the survival and differentiation of NSCs following in vivo application, we assessed the growth of rat iPSC-NSCs in our integrin binding peptide array platform as we previously found several integrin binding peptides in the array system can support better adhesion and growth of rat primary neurons and human iPSCs [22, 24]. This custom-designed integrin-binding array platform contains short synthetic peptides binding to 16 types of integrins that are commonly expressed on almost all cells in vertebrates including α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α6β4, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α_Lβ₂, α_Mβ₂ and α_IIbβ₃. The rat iPSC-NSCs were plated onto the integrin binding peptides array followed by two weeks culturing with the optimized differentiation medium, cells were fixed and immunostained for TUJ1, MAP2 and DAPI. Microscopically, when examining cell grown among the 16 integrin binding peptides coated wells and PLO coated control wells, the best cell growth was observing in the wells coated with α_vβ₈ binding peptide as demonstrated by the number of TUJ1+ or MAP2+ cells as well as the extent of neurite outgrowth (Fig. 6a). Significant higher percentage of relative fluorescent intensity of TUJ1+ cells was found in cells grown in the α_vβ₈ binding peptide coated wells in comparison to all other conditions (Fig. 6b).

Fig. 6. Representative integrin binding peptides in supporting growth of rat iPSC-NSCs derived neurons.

a). The growth of rat iPSC-NSCs derived neurons was assessed by expressing of neuronal markers of Tuj-1 (red) or MAP2 (green) in the integrin array platform. Representative images showed typical Tuj-1 and MAP2 staining patterns in selected integrin-binding peptide coated wells in comparison to poly-l- ornithine (PLO) coated control well. Note the well coated with α_vβ₈ binding peptides provided cells with the best growth both for the cell number and the neurite outgrowth. Scale bar =100μm. b). Quantification analysis of the relative Tuj1+ staining intensity. Significant higher percentage of Tuj1 expression was observed in cells grown on peptide α_vβ₈ in comparison to other integrin binding peptides and PLO control.

Additionally, we assessed the growth of iPSC-NSCs in 3D culture in a PEG-based injectable hydrogel containing α_vβ₈ binding peptide. In this 3D culture, iPSC-NSCs were mixed with 0.4% PEG hydrogel plus α_vβ₈ binding peptide before being plated onto the hydrogel pre-coated wells or control condition. The growth and differentiation of cells were observed after 4 weeks culture with immunostaining of TUJ1 and MAP2. As shown in Fig. 7, cells in the wells containing the α_vβ₈ binding peptide had higher percentage of TUJ1+ cells and bearing longer neurite processes (Fig. 7). Thus, our data suggests that hydrogel combined with the α_vβ₈ binding peptide can provide a better environment for neuronal differentiation and growth for rat iPSC-NSCs in vitro.

Fig. 7. The 3-D culture with α_vβ₈ binding peptide and hydrogel provided an optimal growth environment for neuronal differentiation and growth of rat iPSC-NSCs.

a-j). Representative confocal images of cells grown in 3D culture expressing TUJ1 (red), MAP2 (green) and DAPI (blue). a-d) Cells grown in hydrogel only condition; f-l) Cells grown in hydrogel plus α_vβ₈ binding peptide condition. e and j). Confocal 3-D reconstruction images for better appreciation of cell growth pattern. k). Quantification analysis of neurite length of Tuj1+ cells in the 3-D culture demonstrated that the matrix with hydrogel plus α_vβ₈ binding peptide provided a better support for neurite outgrowth compared to in hydrogel only condition. l). Quantification analysis of the percentage of Tuj1+ cells against the total number of DAPI+ cells in the 3D culture demonstrated that hydrogel plus α_vβ₈ binding peptide supported higher percentage of cells differentiated into neurons in comparison to cells grown in hydrogel only condition. Scale bar =20μm.

4. Discussion

Due to the limited potential of regeneration of adult brain tissue, there is no effective strategy for brain repair following brain injury. In recent years, stem cell transplantation has attracted much attention for neural repair and regeneration owning to the potential of direct cell replacement and/or production of growth promoting factors by the transplanted cells to promote the endogenous tissue repair. However, thus far, neural transplantation has had very little success and encountered many problems such as limited cell availability, poor long term survival and neuronal differentiation and functional integration, risk of tumor formation, ethical issues and immunological rejection [25–28]. Among all these limitations, the donor cell source is a critical issue and many cell types have been assessed in preclinical studies in varying neurological disease models [29]. Recently, the emerging somatic cell derived iPSCs has opened new avenues for neural transplantation as a potential therapy for the injured brain. Theoretically, iPSCs could be derived from patients themselves for autologous transplantation eliminating all issues encountered in neural transplantation. Thus, the prospective of iPSCs for treating neurological diseases is under intense investigation [30]. So far, the iPSCs that have been explored in preclinical studies are mostly derived from human, with smaller percentages of mice origin [31–33]. As rats are a commonly used animal in modeling neurological injuries, it is imperative to assess the compatibility of iPSCs derived from inbred rat for the rat injury models to achieve the goal of autologous neural transplantation. In this study we reported a detailed method of reprogramming rat fibroblasts into iPSCs and differentiation into NSCs. We also assessed their neuronal differentiation and maturation potential as well as the optimal growth conditions for these cells.

Published studies reporting generation of rat iPSCs from the somatic cells are very limited [34–36]. Traditionally iPSCs are generated by co-infection of individual Yamanaka factors to somatic cells which creates the heterogeneous population of iPSCs [37]. To address this issue, we used a cre-excisable polycistronic lentiviral vector system containing all four factors as the stem cell cassette separated by 2A peptides [23, 38]. The use of a polycistronic lentiviral vector system has several benefits including higher reprogramming efficiency, reduced number of viral integrations and the ability to excise the viral transgenes. Removal of transgenes from iPSCs makes them safer for translational application in clinical use because transgenes pose a risk to tumor formation, and constant expression of transgenes adversely affects the pluripotency [20]. Our study demonstrates the efficacy of this strategy for reprogramming rat fibroblasts into iPSCs. Using this approach, we tested the efficacy of reprogramming of rat fibroblasts isolated from the skin of adult Fisher 344 rats and embryonic origin. Though the reprogramming of fibroblasts from adult rats is achievable in our hands, the embryonic fibroblasts had a much higher efficiency to be reprogrammed, which is in agreement with other published studies reporting somatic cell reprogramming [39, 40].

For neural transplantation, the donor cells must be differentiated into neuronal linage to achieve the goal of neural replacement, and NSCs, not the mature neurons, are generally used as the donor cells. Following grafting into the brain, the extent of survival and neuronal differentiation of grafted NSCs is influenced by the microenvironment of the host brain [11, 41]. In the injured brain, tissue damage induced inflammation, glial scar and blood supply paucity impose a hostile focal environment to the survival and differentiation of the grafted cells [42, 43]. For successful neural transplantation, providing the grafts with biomaterial based supporting structure is desirable [44–47]. As a matrix, the ideal biomaterial should promote delivery of therapeutic agents to minimize neuroinflammation and scar formation as well as facilitate the growth and survival of transplanting cells. Before in vivo application, the compatibility of biomaterials for cell delivery is usually assessed in vitro in 3D cultures. Among the biomaterials in 3D culture system, hydrogels are widely used as they can be customized with various combinations of crosslinked fibers, peptides or the growth promoting factors to enhance neurite growth or modulate neuroinflammation [22, 48]. In the current study, we assessed the survival, neuronal differentiation and maturation of rat iPSC-NSCs in 3D culture in a PEG-based hydrogel supplemented with α_vβ₈ integrin binding peptide, which we found effective in promoting neural differentiation and neurite outgrowth of rat iPSC-NSCs under 2D culture. Our previous studies have reported that the α_vβ₈ integrin binding peptide provided superior condition supporting neuronal growth, survival and maturation of rat derived primary neurons [22], as well as vascular formation of primary brain endothelial cells [48], in both 2D and 3D conditions. Here, we have further reported that PEG-based hydrogel in combination with α_vβ₈ integrin binding peptide is conducive for the neuronal differentiation and growth of rat iPSCs-NSCs in vitro. This combinational strategy will be explored in vivo in a rat brain injury model.

Highlights:

• High efficiency reprogramming of rat fibroblasts into pluripotent stem cells

• Generation of neural stem cells and functional matured neurons from rat-iPSCs.

• Synthetic integrin-binding peptides assisted rat-iPSCs neuronal differentiation

• Improved growth and maturation of neurons derived from rat-iPSCs in 3D culture