Aligned Brain Extracellular Matrix Promotes Differentiation and

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Biological and Medical Applications of Materials and Interfaces

Aligned brain extracellular matrix promotes differentiation and myelination of human induced pluripotent stem cell-derived oligodendrocytes Ann-Na Cho, Yoonhee Jin, Suran Kim, Sajeesh Kumar, Heungsoo Shin, Hoon-Chul Kang, and Seung-Woo Cho ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03242 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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ACS Applied Materials & Interfaces

Aligned brain extracellular matrix promotes differentiation and myelination of human induced pluripotent stem cell-derived oligodendrocytes Ann-Na Cho†1, Yoonhee Jin†1, Suran Kim†, Sajeesh Kumar, Heungsoo Shin, Hoon-Chul Kang||, and Seung-Woo Cho†‡§*

† Department ‡ Center

of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

for Nanomedicine, Institute for Basic science (IBS), Seoul 03722, Republic of Korea

§ Yonsei-IBS

Institute, Yonsei University, Seoul 03722, Republic of Korea

 Department

of Bioengineering, Division of Applied Chemical and Bio Engineering,

Hanyang University, Seoul 04763, Republic of Korea || Division

of Pediatric Neurology, Department of Pediatrics, Yonsei University College of

Medicine, Seoul 03722, Republic of Korea

1These

authors contributed equally to this work.

*Corresponding author Prof. Seung-Woo Cho Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea. E-mail: [email protected] 1

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ABSTRACT Myelination by oligodendrocytes (OL) is a key developmental milestone in terms of the functions of the central nervous system (CNS). Demyelination caused by defects in OL is a hallmark of several CNS disorders. Although a potential therapeutic strategy involves treatment with the myelin-forming cells, there is no readily available source of these cells. OL can be differentiated from pluripotent stem cells; however, there is a lack of efficient culture systems that generate functional OL. Here, we demonstrate biomimetic approaches to promote OL differentiation from human induced pluripotent stem cells (iPSCs) and to enhance the maturation and myelination capabilities of iPSC-derived OL (iPSC-OL). Functionalization of culture substrates using brain extracellular matrix (BEM) derived from decellularized human brain tissue enhanced the differentiation of iPSCs into myelinexpressing OL. Co-culture of iPSC-OL with induced neuronal (iN) cells on BEM substrates, which closely mimics the in vivo brain microenvironment for myelinated neurons, not only enhanced myelination of iPSC-OL, but also improved electrophysiological function of iN cells. BEM-functionalized aligned electrospun nanofibrous scaffolds further promoted the maturation of iPSC-OL, enhanced the production of myelin sheath-like structures by the iPSC-OL, and enhanced the neurogenesis of iN cells. Thus, the biomimetic strategy presented here can generate functional OL from stem cells and facilitate myelination by providing brain-specific biochemical, biophysical, and structural signals. Our system comprising stem cells and brain tissue from human sources could help in the establishment of human demyelination disease models and the development of regenerative cell therapy for myelin disorders.

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Key words: human induced pluripotent stem cells, oligodendrocytes, myelination, human brain extracellular matrix, induced neuronal cells, aligned nanofiber

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INTRODUCTION Oligodendrocytes (OL) play a key role in myelination, which is critical for the development and functioning of the central nervous system (CNS). OL produce the protective and insulating lipid-rich myelin sheath, which covers axons in the CNS, and organize the distribution of axonal voltage-gated ion channels, a prerequisite for the conduction of action potentials and trophic support of axons.1-2 Accumulating evidence suggests that demyelination is associated with several neurodegenerative diseases, such as multiple sclerosis, leukodystrophies, multiple system atrophy, amyotrophic lateral sclerosis, and traumatic spinal cord injury.3-4 A cell therapy technique involving promotion of remyelination by transplanting myelin-producing cells, such as OL, represents a promising new strategy to protect and restore axonal integrity and neurological function in CNS harboring demyelinated axons. However, the development of such cell-based therapies has been restricted by the limited availability of human OL.5 The lack of human myelin disorder models due to difficulty in acquisition of human OL has also impeded the discovery of novel therapeutic drugs that can be used for demyelination diseases. Thus, this has given rise to the need for discovery of alternate sources of OL that can be employed for development of human regenerative medicine and demyelination disease modeling. Stem cell technologies, employing human pluripotent stem cells (PSCs), provide efficient strategies to circumvent these current limitations. Human PSCs, including human embryonic stem cells and human induced pluripotent stem cells (iPSCs), represent a valuable source for the generation of myelinogenic OL for cell-replacement therapies and research models.6-7 In particular, OL differentiation of iPSCs derived from patients’ somatic cells could permit basic studies to delineate demyelination disease mechanisms and provide 4


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autologous cells for replacement therapy and patient-specific disease models. Nevertheless, to date, only a couple of differentiation protocols have been established for the generation of human PSC-derived OL,8-10 and currently available protocols are technically challenging, inefficient, and time-consuming and yet do not yield sufficient number of functionally matured OL capable of inducing axonal myelination. To overcome these problems, several types of natural and synthetic biomaterials have been used to promote OL viability, growth, and differentiation by providing adequate biophysical and biochemical support;11-13 however, functional maturation of OL to increase myelin-forming ability needs to be improved further owing to the lack of brain-specific microenvironments. In addition, most of the studies were conducted with non-human primary cells or embryonic stem cells that are less feasible for clinical settings. Accordingly, previous studies could not successfully reconstitute in vivo-like myelination process with functional human OL. Here, we proposed a biomimetic strategy to promote differentiation and maturation of iPSC-OL using brain-mimicking microenvironments reconstituted with extracellular component (brain extracellular matrix derived from decellularized human brain tissue; BEM) and cellular component (induced neuronal cells; iN cells) in brain tissue. To provide biochemical and biophysical support that can facilitate myelination of iPSC-OL, BEM coating and co-culture with iN cells were done on electrospun aligned nanofibrous scaffolds (Figure 1). Subtle differences in extracellular matrix (ECM) compositions from one type of tissue to another can affect cellular interactions in a lineage-specific manner.14-15 Thus, tissuespecific ECM culture system could provide desirable cell-matrix interactions, which facilitate adequate cell growth and maintain their tissue-specific phenotype and function. Our group has previously shown that tissue-specific ECM (e.g. brain, liver, tongue, etc.) can improve the reliability and efficiency of cell growth, stem cell differentiation, and direct 5



reprogramming to specific lineage cells.16-18 Similarly, here BEM-modified substrates promoted generation of a high number of OL, displaying mature phenotype with highly segmented branches, from human iPSCs. Furthermore, the co-culture of iPSC-OL with iN cells, prepared by direct conversion on BEM substrates, facilitated myelin formation and wrapping around axons of the iN cells. As a result, the excitability of iN cells in response to neurotransmitter glutamate was improved. To further increase the contact guidance cues for myelination of iN cells by iPSC-OL, BEM-coated electrospun aligned nanofibrous scaffolds were used for the co-culture of iPSC-OL and iN cells, which not only promoted neurogenesis of iN cells, but also facilitated reconstitution of myelin-like structures along the aligned nanofibers. Our brain-mimicking platform that recapitulates brain-specific microenvironments may be useful for the development of therapeutic strategies and models associated with demyelination diseases.

RESULTS AND DISCUSSION BEM facilitates iPSC differentiation into O4- and myelin basic protein (MBP)-positive OL We induced human iPSCs to differentiate into oligodendrocyte progenitor cells (OPCs) or OL following a previously established protocol with slight modification (Supporting Information Figure S1A).19 After 12 days of OPC induction, cells were positive for oligodendrocyte transcription factor 2 (OLIG2) and SRY (sex determining region Y)-box 10 (SOX10) (Supporting Information Figure S1B). Differentiated OPCs were positive for platelet-derived growth factor receptor alpha (PDGFRα) as well as OLIG2 and SOX10 after 40 days of 6


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induction (Supporting Information Figure S1C). iPSC-OPCs were further differentiated up to day 55 and were used for further experiments. BEM was obtained through decellularization of human brain tissue and subsequent solubilization of decellularized tissue. Our previous mass spectrometric analysis confirmed that BEM obtained by our protocol contained various brain-specific ECM components, including different types of collagen (type I, IV, and VI), glycoproteins (fibronectin, fibrinogen, laminin, and tenascin), and proteoglycans (brevican, neurocan, versican, and heparan sulfate),18 which can provide biochemical and biophysical niches similar to that of native brain tissue. After 55 days of culture, iPSC-OPCs were transferred onto 0.01 mg/mL BEM-coated substrate and cultured for another 14 days. OL differentiation on the BEM substrate was compared with that on the Matrigel- and laminin (LM)-coated substrates in terms of the expression of OL differentiation marker (O4), cellular area, and cellular morphology. The LM coating (0.01 mg/mL) was included as a positive control, as it is known to promote differentiation of OL,20-21 and has been used as a gold standard ECM in OL culture. The BEM group showed the highest number of O4-positive cell population among the groups (Figure 2A, B). Furthermore, the total area of O4-positive cells per image (normalized to Matrigel group) was 1.64 times larger in the BEM group relative to the LM group (Figure 2A, C). Notably, myelin-like membrane sheet structures were observed in the BEM group. iPSC-OPCs on the BEM substrates were cultured for 21 more days and analyzed for myelin basic protein (MBP)-positive cells that represent functional phenotype for myelination (Figure 3). iPSC-OL in the BEM group displayed mature phenotype with majority of populations displaying more than 10 MBP-positive segments (80%), whereas only 10% population of iPSC-OL exhibited more than 10 MBP-positive segments in the LM group (Figure 3A, B). The MBP-positive cell area in the LM and BEM groups was 1.75 ± 0.79 and 7



6.18 ± 3.51 times larger compared to that in Matrigel group, respectively (Figure 3C). These results clearly indicated that BEM promoted OL differentiation of iPSCs and facilitated maturation of OL with MBP-positive morphology and function.

Myelin-like sheath formation by iPSC-OL is enhanced in presence of BEM and coculture with iN cells The effect of BEM on OL-mediated myelination was examined with co-culture of iPSC-OL with iN cells. The iN cells were obtained by direct cellular reprogramming from primary mouse embryonic fibroblasts (pMEF) on the BEM-coated substrates. They were generated by transfecting the genes encoding three key neuronal transcriptional factors (i.e. Brn2, Ascl1, and Myt1l) into pMEF and culturing for 16 days.18 iPSC-OPCs, at day 55 of culture, were transferred onto either LM- or BEM-coated substrate, and co-cultured with iN cells for 5 days. The maturation of iPSC-OL cultured on the BEM substrate was assessed by analyzing the expression of MBP and myelin formation using immunofluorescence staining (Figure 4). Confocal microscopic imaging revealed that iPSC-OL cultured alone on the LM substrate showed immature MBP-positive OL morphology with few segments at day 60 (Figure 4A). iPSC-OL cultured alone on the BEM substrate exhibited a higher number of MBP-positive OL with multipolar morphology. iPSC-OL with multipolar phenotype were also more frequently observed when co-cultured with iN cells on the LM substrate. This result is consistent with the finding from a previous study that reported that co-culture of rat OPCs with dorsal root ganglion (DRG) neurons on the nanofiber scaffolds increased the number of cells with bipolar and multipolar morphologies, suggesting that axonal cues contribute to morphological and phenotypic stabilization of oligodendroglial cells.11 Amongst all the 8


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groups, iPSC-OL co-cultured with iN cells on the BEM substrate showed the most highly mature phenotype with extended membrane sheaths and highly branched processes (Figure 4A). In the BEM group with iN cell co-culture, abundant MBP-mediated processes that interacted with axons and induced ensheathment of axons were more extensive than in other groups (Figure 4A). Unambiguous myelin formation was not observed in the control groups without iN cell co-culture or BEM coating at the same time point. Comparison of myelin formation of iPSC-OL in the iN cell co-culture groups indicated that the percentage of the MBP-positive cells and the contact with iN cells to form myelin-like sheaths significantly increased in the BEM-coating group (Figure 4B-D). Taken together, these observations suggested that brain-mimicking environments reconstituted by cellular (iN cells) and extracellular (BEM) components promoted differentiation and maturation of iPSC-OL, leading to enhanced myelination process.

iPSC-OL cultured on the BEM improved the electrophysiological function of iN cells in response to neurotransmitter The wrapping of myelin sheath around axons by OL serves as a protective layer for axons and facilitates increased conduction velocity of electrical impulse.22 Hence, the neurotransmitter response of iN cells co-cultured with iPSC-OL was assessed by fluorescence imaging using a calcium influx indicator, Fluo-4-AM (Figure 5). Upon exposure to a neurotransmitter (glutamate), the population of glutamate-responsive iN cells was greater in the co-culture group of iPSC-OL and iN cells on the BEM substrate (Co-culture/BEM group) compared to other control groups without BEM coating or iPSC-OL co-culture (iN cell only/LM, Co-culture/LM, and iN cell only/BEM groups) (Figure 5A). Quantification of the 9



change in the florescence intensity of Fluo-4-AM in cell population indicated that iN cells cocultured with iPSC-OL on the BEM substrate exhibited the most prominent change in cytoplasmic calcium influx compared to other control groups (Figure 5B). These data suggested that co-culture with iPSC-OL in the presence of BEM improved the neurotransmitter response and excitability of the iN cells, and in turn, their electrophysiological functionality, probably due to enhanced myelination of iN cells by iPSCOL and BEM. Thus, engineering the interfaces between cells and culture substrates with brain-specific ECM components could be a potential strategy to enhance neuronal network formation and electrical signal transmission.

BEM-modified aligned nanofibers further promote neuronal maturation and formation of myelin-like structures Biophysical cues to facilitate contact guidance can promote neurogenesis and myelination of stem cells. Here, aligned nanofibrous scaffolds modified with BEM were utilized to increase brain-mimetic biophysical contact guidance for iN cells and iPSC-OL. Electrospun nanofibrous scaffolds exhibit several key properties that are advantageous for neural tissue engineering, including the large surface-to-volume ratio, a high degree of porosity, and the hierarchical structure of the native ECM.23 Aligned nanofibers with an average diameter of 200-300 nm were fabricated from poly(ε-caprolactone) (PCL), a clinically approved biocompatible and biodegradable polyester,24 using electrospinning technique. Axons and dendrites of neurons in both central and peripheral nervous systems have highly elongated, thin cellular structures that often show distinct patterns of alignment.25 Hence, the aligned nanofibers might provide structural guidance for growth of iN cells, while they could also act 10


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as pseudo-axonal scaffolds which facilitate formation of myelin-like segments for iPSC-OL. The fiber size ranging from 200-400 nm has been reported to be favorable for OL culture, probably due to the morphological resemblance to axons.13 BEM coating on the aligned PCL nanofibers further mimics the topographical and biochemical cues provided by the brain ECM in vivo. Scanning electron microscopy confirmed the deposition of BEM on cover-glass and aligned fibers (Figure 6A). Live/Dead staining assay showed that iPSC-OL, grown on the BEM-coated aligned PCL nanofibers, were highly viable and aligned along the fiber structures (Figure 6B). In the co-culture of iPSC-OL and iN cells, BEM-functionalized aligned nanofibrous scaffolds (200-300 nm in diameter) were employed to provide brain-mimetic biochemical and structural cues for promoting neurogenesis of iN cells and myelination of iPSC-OL. The iPSC-OL were cultured alone on BEM-coated nanofibrous scaffolds (iPSC-OL/BEM fiber), and co-cultured with iN cells on BEM-coated cover-glass (Co-culture/BEM glass) or nanofibrous scaffolds (Co-culture/BEM fiber) for 5 days. In co-culture conditions, a higher proportion of iPSC-OL exhibited myelin wrapping around axons of iN cells when they were cultured on BEM-coated nanofibers, compared to those cultured on BEM-coated cover-glass (Figure 6C). Comparison of segmentation in all conditions indicated that BEM-coated nanofibrous scaffolds efficiently induced formation of highly segmented branches in iPSCOL (Figure 6D). A higher degree of extension of MBP-positive multiple segmented branches from iPSC-OL was seen along the nanofibers in the co-culture/BEM fiber group with some of these extensions wrapping around iN cells grown along the nanofibers (Figure 6D). These results demonstrated that BEM-modified nanofibers facilitated formation of myelin sheathlike structures by increasing contact guidance for both iPSC-OL and iN cells.

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To assess the effect of BEM-modified aligned nanofiber structures on differentiation and maturation of iPSC-OL on a genetic level, quantitative real time-polymerase chain reaction (qPCR) for oligodendroglial lineage markers was conducted. iPSC-OL cultured on the BEM-coated aligned PCL nanofibrous scaffolds exhibited higher expression of MBP (2.67  0.39), 2,3-cyclic nucleotide 3-phosphodiesterase (CNP) (1.38 ± 0.08), and oligodendrocyte transcription factor 1 (OLIG1) (1.16 ± 0.05) compared to the cells cultured on BEM-coated cover-glass (Figure 6E). Similar gene expression profiles were observed in co-culture setting where MBP expression was almost 2-fold higher in the BEM fiber group than that in the BEM glass group (1.93 ± 0.18) (Figure 6F). The gene expression level of other oligodendroglial lineage markers, CNP (1.34 ± 0.08) and OLIG1 (1.18 ± 0.04), was also increased in the BEM fiber group compared to that in the BEM glass group (Figure 6F). The gene expression level of MBP in iPSC-OL grown on the BEM-coated substrates was compared to that in the cells grown on conventional ECM-coated substrates (Matrigel and LM) after 7 days of culture (Figure 6G). Significantly higher expression level of MBP was observed in co-culture/BEM glass, iPSC-OL/BEM fiber, and co-culture/BEM fiber groups, when compared with iPSC-OL/Matrigel glass, iPSC-OL/LM glass, and co-culture/LM glass groups (Figure 6G). The highest MBP expression level was detected in the iPSC-OL/BEM fiber and co-culture/BEM fiber groups, suggesting that nanofibrous structures functionalized using BEM may be an important factor influencing the MBP expression in iPSC-OL. The analysis of gene expression of neuronal marker, class III beta-tubulin (Tuj1), in iN cells revealed that co-culture of iPSC-OL and iN cells on BEM and/or aligned nanofiber significantly increased the expression of Tuj1 (Figure 6H). The highest Tuj1 expression was found when iN cells were co-cultured with iPSC-OL on the BEM-coated aligned nanofiber scaffolds (3.46 ± 0.57 in the LM/fiber group, 4.46 ± 0.86 in the BEM/glass group, and 5.33 ± 12


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1.04 in the BEM/fiber group, compared to the LM/glass group), suggesting that aligned nanofibrous structures engineered with BEM components promoted both differentiation of iPSC-OL and neuronal differentiation of iN cells. Biochemical compositions of the brain ECM play a key role during oligodendrogenesis.20, 26-28 BEM contains a variety of ECM molecules18 that were previously reported to influence OL maturation and myelination. For example, laminin α2 promotes OPC survival and maturation into OL by promoting myelin membrane formation.20, 28 Hence, BEM, which was coated even on the cover-glass, could provide biochemical signals necessary for oligodendrogenesis, resulting in enhanced OL maturation and myelination (Figure 6D, G). Electrospun aligned nanofibrous scaffold can provide biophysical cues for iPSC-OL. A previous study demonstrated that OPCs cultured on polymer nanofibers tightly associate with the fibers and proliferate until reaching a critical density before differentiation, representing the processes observed when OPCs are co-cultured with neuronal axons.11 The fibrous structures seem to provide the axon-like signals for myelin compaction and establishment of the appropriate wrapping of membrane in the myelin sheath.11 Thus, similar to these previous observations, the culture of iPSC-OPCs on our aligned PCL nanofibrous scaffolds may be able to promote maturation and myelination of iPSC-OL even without BEM modification. In this study, we have, for the first time, demonstrated that the maturation and myelination of OL, derived from clinically relevant type of human stem cells (i.e. human iPSCs), could be modulated by providing human brain tissue-mimicking biochemical and biophysical microenvironments. Several studies examined the effects of biophysical and mechanical elements on differentiation and myelination of OL or OPCs. Previously, OPC 13



culture platform based on electrospun nanofibers was employed as a neuron-free myelination model to manipulate and investigate the biophysical factors of axonal-oligodendroglial interactions.11 Primary rat OPCs cultured on the poly(L-lysine) (PLL)-coated electrospun nanofibrous scaffolds with varied fiber diameters in the range of 0.2-4.0 µm exhibited different levels of ensheathment and wrapping of the fibers depending on the fiber diameter.11 Another study presented an artificial axon model, based on 3D-printed polymer fibers, for myelination assay.12 The developed platform allowed for direct visualization and quantification of the effects of geometric, mechanical, and surface properties of axon-like fiber structures on differentiation and myelin wrapping of OPCs. Varying stiffness (0.4 kPa versus 140 kPa), diameter (10 µm versus 20 µm), and ligand coating (poly(D-lysine) versus LM) of 3D-printed fibers, as microenvironment parameters for myelination, altered artificial axon wrapping of primary rat OPCs.12 Although these studies could provide effective myelination modeling to address the important roles of biophysical factors in developmental myelination and remyelination processes, human neuronal tissue-specific components (e.g. human brain ECM) need to be incorporated into the models to better recapitulate human myelination process in vitro. In addition, because most of previous studies on engineering OL or OPC culture were demonstrated with animal primary cells and immortalized cell lines, their systems may not be applicable to clinically-relevant human cells. Our approach to reconstitute brain-mimetic cellular and extracellular components guided by the scaffolds would be useful for cell transplantation and tissue regeneration to address traumatic injuries and severe demyelination disorders with severely damaged neuronal and OL populations. Actually, biomaterials applied to reconstitute a brain-like microenvironment in our study (decellularized tissue matrix and PCL nanofibrous scaffold) have been used in a variety of tissue engineering and regenerative medicine approaches in preclinical and clinical studies. 14


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PCL scaffolds are biocompatible and biodegradable synthetic materials that have been already approved for clinical applications by Food and Drug Administration (FDA). Decellularized tissue matrix derived from organs and tissues has been tested for therapeutic clinical applications to regenerate various tissues such as trachea,29-32 heart values,33-37 cornea,38 and bladder.39-40 Several products made of acellular dermal matrix including an FDA-approved one, PriMatrix, are also available for clinical application to regenerate skin tissue in the management of skin ulcers, trauma wounds, surgical wounds, and second degree burns. Since an aligned, brain-mimetic microenvironment created by combining these two biomaterial platforms (decellularized matrix and PCL scaffolds) facilitates generation of functional OL from stem cells and interaction between OL and neurons, our strategy based on biomaterials approved for human applications is expected to increase clinical potential of tissue-engineered constructs capable of promoting myelination in demyelination diseases. Although decellularized tissue matrix and PCL scaffolds are feasible for translation to clinical settings and their production is scalable, an optimized protocol for generating the large number of OL would also be critical to potentiate the translational and clinical applications of our approach. The application of the device platforms prepared with various microfabrication techniques (e.g. microfluidics, microwell, etc.) could also be considered at the stages of OPC expansion and suspension culture of OPCs, which are known to determine OPC differentiation capacity to OL, thereby facilitating OL differentiation of human stem cells and scale-up the production of OL.41 Decellularized tissue matrix has a great potential for regenerative medicine, but batch-to-batch variations in BEM might restrict its use in clinical treatments. Tissue sources and storage conditions before being processed for decellularization influence the ECM compositions and quality of BEM, resulting in batch‐to‐batch difference. In our previous 15



study, mass spectroscopic analysis indeed revealed that ECM compositions in human BEM vary depending on the sources (brain regions, age, gender, etc.).18 Thus, control of the amount and concentrations of ECM components between different BEM batches would be an important challenge for further therapeutic applications. Although the type and quantity of ECM components are heterogeneous in different BEM batches, BEM may be able to consistently provide proteoglycan-enriched, brain ECM microenvironments as the content of fibrotic ECM proteins (e.g. collagen, fibronectin) is generally low while proteoglycans (e.g. neurocan, versican, heparan sulfate) are relatively abundant in brain tissue.42-43 Consequently, different BEM batches consistently promoted neuronal reprogramming at similar levels, indicating that there was a marginal batch variation in the key properties and functionality of human BEM for reprogramming in spite of a variation in ECM compositions.18 However, it is obvious that universally accepted quality control for tissue sourcing and storage of BEM would prove beneficial in creating a more reliable system for OL differentiation and myelination. In order to minimize batch-to-batch difference, decellularization process should be conducted with strict guidelines and the mixture of the samples from different batches could be used to make the ECM compositions less heterogeneous. The cell sources used in our study (human iPSC-derived OL and reprogrammed iN cells) have also similar concerns caused by batch-to-batch variation in cell population. Generation of OL from human iPSCs provides an attractive alternative to overcome the limited access to primary tissues and is expected to offer significant advantages for exploring demyelination-related pathogenesis and regenerative medicine. However, heterogeneity of human iPSC-derived OL due to lengthy differentiation protocols (more than 50~70 days) and low OL differentiation efficiency result in significant batch-to-batch difference. As iN cells are artificially reprogrammed from fibroblasts by genetic engineering, they are under 16


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different reprogramming status, leading to heterogeneity in terms of the degree of differentiation and maturation. Therefore, low regenerative efficacy and potential side effects due to such heterogeneity and batch-to-batch variation in cell sources need to be addressed to improve regenerative efficiency of our system for myelination. In addition to optimization of differentiation and reprogramming protocols, robust selection strategy to highly specific lineage markers would be considered to guarantee homogenous cell population with stable phenotypes and high functionality.

CONCLUSIONS In summary, we recapitulated brain-mimetic microenvironments with cellular (iN cells) and extracellular (BEM) components combined with structural cues (aligned nanofibers) to provide optimal biochemical, biological, and physical milieu relatively similar to in vivo brain niches. To the best of our knowledge, our study is the first report demonstrating a bioinspired strategy to develop functional biomaterials that can promote differentiation, maturation, and myelination of human iPSC-OL. The approach to reconstitute such brainspecific factors on a single biomaterial platform would be invaluable and highly beneficial for cell therapy, tissue engineering, and disease modeling. Our strategy may potentially eliminate the use of exogenous factors, such as viral gene vectors, growth factors, and small molecular weight drugs for enhancing differentiation and myelination of OL from human stem cells. We envision that our platform may serve as a powerful tool for the development of novel therapies against CNS-related disorders and injuries.

MATERIALS AND METHODS 17



iPSC culture and OL differentiation. Human iPSC cell line (WT3) was kindly provided by Yonsei University College of Medicine and the use of human iPSCs was approved by the Institutional Review Board (IRB) of Yonsei University (Permit Number: 1040917-201510BR-229-01E). The culture conditions for the maintenance of iPSCs (passages 29-45) were described previously.44-45 Briefly, iPSCs were cultured on a feeder cell layer, consisting of STO fibroblasts (American Type Culture Collection, Manassas, VA) treated with 10 μg/mL mitomycin C (Sigma-Aldrich, St. Louis, MO), in iPSC maintaining medium, consisting of Dulbecco’s modified Eagle medium/nutrient mixture F12 (DMEM/F12, Thermo Fisher Scientific, Gaithersburg, MD), 20% (v/v) Knockout Serum Replacement (KSR, Thermo Fisher Scientific), 1% (v/v) penicillin-streptomycin (P/S, Thermo Fisher Scientific), 1× nonessential amino acid (NEAA, Thermo Fisher Scientific), 0.1 mM β-mercaptoethanol (SigmaAldrich), and 10 ng/mL basic fibroblast growth factor (bFGF, R&D System, Minneapolis, MN). OL differentiation from iPSCs was conducted using a previously reported protocol with minor modifications (Supplementary Figure S1A).19 Using 2 mg/mL (w/v) collagenase type IV (Thermo Fisher Scientific), iPSCs were passaged onto Matrigel (Corning, Corning, NY)-coated culture dish (TPP, Trasadingen, Switzerland) in a feeder-free iPSC maintaining medium (Essential 8 medium, Thermo Fisher Scientific). The next day, iPSC maintaining medium was replaced with neural induction medium composed of DMEM/F12, 1% (v/v) P/S, 1× NEAA, 1× GlutaMax (Thermo Fisher Scientific), 0.1 mM β-mercaptoethanol, 25 μg/mL (w/v) insulin (Sigma-Aldrich), 100 nM all-trans retinoic acid (RA, Sigma-Aldrich), 10 μM SB431542 (Sigma-Aldrich), and 5 μM Dorsomorphin (Sigma-Aldrich). The medium was exchanged every day for 8 days. Then, the cells were committed to neuronal lineage by culturing the cells in N2 medium, consisting of DMEM/F12, 1% (v/v) P/S, 1× NEAA, 1× 18


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GlutaMax, 0.1 mM β-mercaptoethanol, 1× N2 supplement (Thermo Fisher Scientific), 100 nM RA, and 1 μM smoothened agonist (SAG, EMD Millipore, Billerica, MA) for 4 days. At day 12 of the culture, iPSC colonies were mechanically dissociated into smaller pieces using a modified Pasteur pipet and enzymatically detached by treatment with 2 mg/mL (w/v) collagenase type IV. The cells were then transferred onto petri dish (SPL, Pocheon, Korea) and cultured in N2B27 medium, consisting of DMEM/F12, 1% (v/v) P/S, 1× NEAA, 1× GlutaMax, 0.1 mM β-mercaptoethanol, 25 μg/mL (w/v) insulin, 1× N2 supplement, 1× B27 supplement (Thermo Fisher Scientific), 100 nM RA, and 1 μM SAG for 8 days to induce early OPCs. At day 20 in the culture, the medium was changed to N2B27 medium supplemented with 10 ng/mL recombinant human platelet-derived growth factor-AA (PDGF-AA, R&D Systems), 5 ng/mL recombinant human hepatocyte growth factor (HGF, Peprotech, Rocky Hill, NJ), 10 ng/mL recombinant human insulin-like growth factor-I (IGF-I, R&D Systems), 10 ng/mL Neutrophin 3 (NT3, EMD Millipore), 25 μg/mL (w/v) insulin, 60 ng/mL 3,3,5-Triiodo-L-thyronine (T3, Sigma-Aldrich), 100 ng/mL biotin (Sigma-Aldrich), and 1 μM N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (cAMP analog, Sigma-Aldrich), and the cells were further cultured in suspension for 10 days. At day 30, the OPC spheroids were transferred to Matrigel-coated culture dish and cultured in OL differentiation medium, consisting of N2B27 medium supplemented with 10 mM HEPES (Sigma-Aldrich), 25 μg/mL (w/v) insulin, 60 ng/mL (w/v) T3, 100 ng/mL (w/v) biotin, 1 μM cAMP, and 20 μg/mL (w/v) L-Ascorbic acid (AA, Sigma-Aldrich) until further use or analysis.

iN cell generation. Primary mouse embryonic fibroblasts (pMEF) were isolated from ICR mouse embryos (E13.5, Orientbio, Seongnam, Gyonggi-do, Korea) as previously reported.18, 19



46

In brief, pMEF (passage 1) were cultured on 0.2% (w/v) gelatin (Sigma-Aldrich)-coated

substrates in high-glucose DMEM (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific), 50 units (v/v) of P/S, 1× NEAA, and 1× β-mercaptoethanol. iN cells were generated as previously reported.18, 46 Briefly, pMEF were transfected with Brn2-, Ascl1-, and Myt1l-encoding plasmids (Addgene, Watertown, MA) by electroporation (2.4 μg/105 cells, Neon, Thermo Fisher Scientific), and seeded on 0.02 mg/mL poly-L-lysine (PLL, Sigma-Aldrich) and 0.01 mg/mL laminin (LM, Thermo Fisher Scientific)-coated cell culture plate. After 2 days of initial seeding, the plasmids encoding three transcriptional factors were re-transfected into the cells (1 μg/105 cells) using Lipofectamine 2000 (Thermo Fisher Scientific). The fibroblast growth medium was replaced with neural induction medium consisting of DMEM/F12, 1× NEAA, 50 units (v/v) of P/S, N2 supplement, 10 ng/mL bFGF, and 10 ng/mL epidermal growth factor (EGF, Peprotech). Subsequently, the cells were further cultured in a neural differentiation medium containing DMEM/F12:Neurobasal (3:1, Thermo Fisher Scientific), 1× NEAA, 1× GlutaMax, 1× N2, 1× B27, and 10 ng/mL brain-derived neurotrophic factor (BDNF, Peprotech) until further use or analysis.

Fabrication of PCL aligned nanofibrous scaffolds. PCL nanofibers were produced using electrospinning method. PCL solution (Sigma-Aldrich) at 8% (w/v) concentration was prepared in a solution containing tetrahydrofuran (Duksan, Ansan, Korea) and dimethylformamide (Duksan) in 8:2 ratio. The polymer solutions fed into a blunted stainlesssteel needle attached to an infusion pump (SHB366, Sckjmoter, China). The solution was electrospun onto an aluminum foil-covered collector at a rotation rate of 100 rpm. The solution flow rate, applied voltage, and spinning time were fixed to 2 mL/h, 11 kV, and 5 h, 20


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respectively, using a high-voltage power supply from NanonC (Seoul, Korea). To evaporate the solvent, the PCL nanofibrous scaffolds were dried at 40 °C overnight in a vacuum oven. The scaffolds were sterilized by soaking in 70% ethanol and dried under ultraviolet-light illumination.

Decellularization of human brain tissue and BEM coating. Human brain tissues were collected from occipital and parietal regions, and used with the approval of the Institutional Review Board (IRB, 4-2014-0769) of Yonsei University College of Medicine. Decellularization of human brain tissues was performed as previously reported.18 Decellularized brain tissues were lyophilized and solubilized by pepsin treatment (SigmaAldrich, 4 mg/mL pepsin per 40 mg/mL tissue in 0.01 M HCl) under stirring for 48 h at room temperature. The resultant BEM solution was used to coat the cover-glass or electrospun PCL scaffolds. For BEM coating, PLL solution (0.02 mg/mL) was first coated on the substrates by incubation for 90 min at 37 °C and washed twice with phosphate buffered saline (PBS). Then, BEM solution was diluted to 0.01 mg/mL in 0.02 M AcOH (Sigma-Aldrich), incubated on the PLL-coated substrates for 120 min at 37 °C, and washed with PBS twice before use.

Co-culture of iPSC-OL and iN cells. iN cells were harvested at day 16 and seeded at a density of 0.5  105 cells/cm2 on 0.02 mg/mL PLL and 0.01 mg/mL BEM- or 0.01 mg/mL LM-coated substrates. After three days of iN cell seeding, iPSC-OPCs were detached at day 55 in differentiation using Accutase (Thermo Fisher Scientific) and seeded at a density of 2.6  105 cells/cm2, and co-cultured with iN cells in OL differentiation medium, consisting of N2B27 medium, supplemented with 10 mM HEPES, 25 μg/mL (w/v) insulin, 60 ng/mL 21



(w/v) T3, 100 ng/mL (w/v) biotin, 1 μM cAMP, and 20 μg/mL (w/v) AA until further analysis.

Immunofluorescence staining. The cells were fixed with 10% formalin solution (SigmaAldrich) for 10 min at room temperature. The fixed cells were permeabilized by adding 0.1% Triton X-100 (Sigma-Aldrich), diluted in PBS, for 10 min at room temperature. Subsequently, blocking was performed by incubating the samples with 5% bovine serum albumin (MP Biomedicals, Santa Ana, CA) and 2% horse serum (Thermo Fisher Scientific) for 1 h at room temperature. The samples were then incubated with primary antibodies for overnight at 4 °C: Olig2 (1:1000, #AB9610, EMD Millipore), SOX10 (1:1000, #AF2864, R&D Systems), PDGFRα (1:200, #sc21789, Santa Cruz Biotechnology, Dallas, TX), O4 (1:1000, #MAB345, EMD Millipore), MBP (1:1000, #ab7349, Abcam, Cambridge, UK), and Tuj1 (1:500, #801213, Biolegend, San Diego, CA). After washing with PBS thrice, the samples were incubated with Alexa Fluor 488 or Alexa-Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific) diluted in PBS for 1 h at room temperature, and subsequently, washed twice with PBS. Nuclei were counterstained with 4,6-diamidino-2phenylindole (DAPI, TCI America, Portland, OR) and washed with PBS. The samples were mounted with fluorescent mounting medium (Vector laboratories, Burlingame, CA) and visualized on a confocal microscope (LSM 880, Carl Zeiss, Jena, Germany). Image quantification analysis was conducted for O4 and MBP based on their immunofluorescence staining data. Marker positive cells were manually counted per field image and positive area was measured per field image using ImageJ 1.49v software (National Institutes of Health, Bethesda, MD).

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Calcium imaging. Cells were incubated with the cell permeable Ca2+ indicator Fluo-4-AM (Thermo Fisher Scientific) at a concentration of 2 μM for 30 min at 37 °C. Cytosolic calcium dynamics were visualized by time-lapse imaging using a confocal microscope (LSM 880, Carl Zeiss) after adding 100 µM glutamate (Sigma-Aldrich). Zen software (Carl Zeiss) was used to determine the fluorescence intensity over a fixed area using Fluo-4-AM as an indicator to determine the presence of cytosolic Ca2+.

Quantitative real-time polymerase chain reaction (qPCR) analysis. Total RNA was isolated from cells using RNA extraction kit (Takara Bio Lnc., Kusatsu, Shiga, Japan) according to the manufacturer’s protocol. cDNA was generated from isolated RNA by reverse transcription using the PrimeScript II first strand cDNA synthesis kit (Takara Bio Lnc.). qPCR analyses were performed on StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA) with the TaqMan Fast Universal Master Mix system (Applied Biosystems). Gene expression was quantified using TaqMan Gene Expression Assays (Applied Biosystems) for each target (human MBP: Hs00921945_m1, human CNP: Hs00263981_m1, human OLIG1: Hs00744293_s1, mouse Tuj1: Mm00727586_s1). The relative gene expression values were calculated by the comparative Ct method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene (human GAPDH: Hs02786624_g1, mouse Gapdh: Mm99999915_g1).

Statistical analysis. All data were presented as mean ± standard deviation (SD) for n  3. A two-tailed unpaired Student’s t-test (GraphPad, San Diego, CA) was used to assess statistical 23



significance of the results (p < 0.05 and p < 0.01 were considered to be statistically significant).

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Immunofluorescence images of human induced pluripotent stem cell-derived oligodendrocyte progenitor cells (iPSC-OPCs) (doc)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Seung-Woo Cho: 0000-0001-8058-332X Ann-Na Cho: 0000-0003-0047-8867 Yoonhee Jin: 0000-0002-1263-9405 Suran Kim: 0000-0002-7981-5573 Author Contributions A-N.C. and Y.J. contributed equally to this paper. Notes The authors declare no competing financial interest. 24


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ACKNOWLEDGMENTS This research was supported by grants (2015M3A9B4071076, 2016M3C9A4921712, and 2017R1A2B3005994) of the National Research Foundation of Korea (NRF) funded by the Korean government, the Ministry of Science and ICT (MSIT), Republic of Korea. This work was also supported by the grant from the Institute for Basic Science (IBS-R026-D1).

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FIGURES AND FIGURE LEGENDS

Figure 1. Schematic illustration of co-culture of human induced pluripotent stem cellderived oligodendrocytes (iPSC-OL) with induced neuronal (iN) cells derived from primary fibroblasts in a brain-mimetic microenvironment. iPSC-OL were co-cultured with iN cells on aligned nanofibrous scaffolds functionalized with decellularized human brain extracellular matrix (BEM).

29



Figure 2. Enhancement of oligodendrocyte (OL) differentiation of human induced pluripotent stem cells (iPSCs) cultured on human brain extracellular matrix (BEM)coated substrates. (A) Immunostaining analysis of iPSC-OL cultured for 14 days on BEM-, Matrigel-, and laminin (LM)-coated substrates with oligodendrocyte marker, O4 (day 69 post-OL differentiation of iPSCs) (scale bars = 100 μm). Quantification of (B) O4-positive cells (N = 5) and (C) O4-positive cell area in iPSC-OL cultured for 14 days on BEM-, Matrigel-, and LM-coated substrates (N = 13-14) (*P < 0.05 and **P < 0.01 versus Matrigel group, ++P < 0.01 versus LM group).

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Figure 3. Maturation of human induced pluripotent stem cell-derived oligodendrocytes (iPSC-OL) into myelin basic protein (MBP)-positive cells after culturing on human brain extracellular matrix (BEM)-coated substrates. (A) Immunofluorescence staining of iPSC-OL against mature oligodendrocyte marker, MBP, after 21 days of culture on BEM-, Matrigel- and laminin (LM)-coated substrates (day 76 post-OL differentiation of iPSCs) (scale bars = 50 μm). Quantification of (B) myelin segment number per MBP-positive cell and (C) MBP-positive total cell area per image (N = 4-6; *P < 0.05 versus Matrigel group and +P < 0.05 versus LM group).

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Figure 4. Enhanced myelination of human induced pluripotent stem cell-derived oligodendrocytes (iPSC-OL) cultured on human brain extracellular matrix (BEM) and co-cultured with induced neuronal (iN) cells. (A) Immunofluorescence staining analysis of iPSC-OL only, iN cell only, and co-culture groups cultured on laminin (LM)-, and BEMcoated substrates for 5 days with mature OL marker myelin basic protein (MBP) and neuronal marker class III beta-tubulin (Tuj1) (scale bar = 100 μm). Image-based quantification of (B) the percentage of MBP-positive cells and (C) the percentage of MBPpositive cells that show wrapping around the axons of iN cells (N = 5; **P < 0.01 versus LM group). (D) Representative immunofluorescence images of the co-culture group (iPSC-OL and iN cells) cultured on BEM substrates after 5 days of culture. Yellow boxes indicate magnified regions. MBP-positive iPSC-OL exhibited wrapping around the axons of iN cells to form myelin (scale bars = 50 μm for the white lines and 20 μm for the yellow lines). 32


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Figure 5. Enhanced calcium channel activation of induced neuronal (iN) cells by human brain extracellular matrix (BEM) in co-culture with human induced pluripotent stem cell-derived oligodendrocytes (iPSC-OL). (A) Radiometric calcium imaging of Fluo-4AM-loaded iN cells only or iN cells co-cultured with iPSC-OL on laminin (LM)- or BEMcoated substrates (scale bar = 100 μm). (B) Quantification of the peak fluorescence intensity of cells after treatment with 100 μM glutamate (N = 30; **P < 0.01 versus iN cells cultured on LM, +P < 0.05 and ++P < 0.01 versus co-culture on LM, and #P < 0.05 versus iN cell cultured on BEM).

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Figure 6. Enhanced maturation and myelination of human induced pluripotent stem cell-derived oligodendrocytes (iPSC-OL) grown on human brain extracellular matrix (BEM)-modified electrospun aligned nanofibrous polycaprolactone (PCL) scaffolds. (A) Scanning electron microscopy (SEM) images of laminin (LM)- or BEM-coated cover-glass and electrospun aligned nanofibrous scaffold (scale bars = 100 nm for the black lines and 2 μm for the white lines). (B) Viability and alignment analysis of iPSC-OL cultured on BEMcoated aligned nanofibrous PCL meshes at day 3 using Live/Dead assay (scale bar = 100 μm). (C) Quantification of myelin-wrapped axons of induced neuronal (iN) cells grown with iPSC-OL on the BEM-coated scaffold (N = 4; **P < 0.01 versus cover-glass (Glass) group). (D) Immunostaining analysis against OL marker myelin basic protein (MBP) and neuronal 34


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marker class III beta-tubulin (Tuj1), to analyze myelination of iPSC-OL cultured alone or cocultured with iN cells on BEM-coated cover-glass or aligned nanofibrous PCL scaffolds (scale bars = 50 μm for white lines and 20 μm for yellow lines). Comparison of gene expression levels of OL lineage markers in iPSC-OL when (E) cultured alone or (F) cocultured with iN cells on BEM-coated cover-glass or aligned nanofiber PCL scaffolds (N = 4; **P < 0.01 versus Glass group). (G) Comparison of expression levels OL marker, MBP, of iPSC-OL cultured alone or co-cultured with iN cells on LM- or BEM-coated cover-glass or aligned nanofibrous PCL scaffolds (N = 4; *P < 0.05, **P < 0.01 versus iPSCOL/Matrigel/Glass group, +P < 0.05 and ++P< 0.01 versus iPSC-OL/LM/Glass group, ##P < 0.01 versus co-culture/LM/Glass group, ^P < 0.05 versus iPSC-OL/BEM/Glass group). (H) Comparison of expression levels of neuronal marker, Tuj1, of iN cells co-cultured with iPSCOL. These cells were cultured on either LM- or BEM-coated cover-glass or nanofiber scaffolds (N = 4; **P < 0.01 versus LM/Glass group, +P < 0.05 versus LM/Fiber group, and #P < 0.05 versus BEM/Glass group).

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Aligned Brain Extracellular Matrix Promotes Differentiation and

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