Yuanwei Yan

Neural differentiation from pluripotent stem cells: The role of natural and synthetic extracellular matrix.

Neural cells differentiated from pluripotent stem cells (PSCs), including both embryonic stem cells and induced pluripotent stem cells, provide a powerful tool for drug screening, disease modeling and regenerative medicine. High-purity oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs) have been derived from PSCs recently due to the advancements in understanding the developmental signaling pathways. Extracellular matrices (ECM) have been shown to play important roles in regulating the survival, proliferation, and differentiation of neural cells. To improve the function and maturation of the derived neural cells from PSCs, understanding the effects of ECM over the course of neural differentiation of PSCs is critical. During neural differentiation of PSCs, the cells are sensitive to the properties of natural or synthetic ECMs, including biochemical composition, biomechanical properties, and structural/topographical features. This review summarizes recent advances in neural differentiation of human PSCs into OPCs and NPCs, focusing on the role of ECM in modulating the composition and function of the differentiated cells. Especially, the importance of using three-dimensional ECM scaffolds to simulate the in vivo microenvironment for neural differentiation of PSCs is highlighted. Future perspectives including the immediate applications of PSC-derived neural cells in drug screening and disease modeling are also discussed.

Differential effects of acellular embryonic matrices on pluripotent stem cell expansion and neural differentiation.

Extracellular matrices (ECM) derived from pluripotent stem cells (PSCs) provide a unique tissue microenvironment that can direct cellular differentiation and tissue regeneration, and rejuvenate aged progenitor cells. The unlimited growth capacity of PSCs allows for the scalable generation of PSC-secreted ECMs. Therefore, the derivation and characterization of PSC-derived ECMs is of critical importance in drug screening, disease modeling and tissue regeneration. In this study, 3-D ECMs were generated from decellularized undifferentiated embryonic stem cell (ESC) aggregates (AGG), spontaneously differentiated embryoid bodies (EB), and ESC-derived neural progenitor cell (NPC) aggregates. The capacities of different ECMs to direct proliferation and neural differentiation of the reseeded mouse ESCs and human induced pluripotent stem cells (iPSCs) were characterized. Proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed protein expression profiles that reflected distinct niche properties for each tested ECM group. The reseeded mouse ESCs and human iPSCs responded to different types of ECMs with different cellular phenotypes. Cells grown on the AGG-ECM displayed high levels of pluripotent markers Oct-4 and Nanog, while the cells grown on the NPC-ECM showed increased expression of neural marker β-tubulin III. The expression levels of β-catenin were high for cells grown on the AGG-ECM and the EB-ECM, but reduced in cells grown on the NPC-ECM, indicating a possible role of Wnt/β-catenin signaling in the cell-matrix interactions. This study demonstrates that PSC-derived ECMs can influence stem cell fate decisions by providing a spectrum of stem cell niche microenvironments during tissue development.

Crosslinking of extracellular matrix scaffolds derived from pluripotent stem cell aggregates modulates neural differentiation

UNLABELLED At various developmental stages, pluripotent stem cells (PSCs) and their progeny secrete a large amount of extracellular matrices (ECMs) which could interact with regulatory growth factors to modulate stem cell lineage commitment. ECMs derived from PSC can be used as unique scaffolds that provide broad signaling capacities to mediate cellular differentiation. However, the rapid degradation of ECMs can impact their applications as the scaffolds for in vitro cell expansion and in vivo transplantation. To address this issue, this study investigated the effects of crosslinking on the ECMs derived from embryonic stem cells (ESCs) and the regulatory capacity of the crosslinked ECMs on the proliferation and differentiation of reseeded ESC-derived neural progenitor cells (NPCs). To create different biological cues, undifferentiated aggregates, spontaneous embryoid bodies, and ESC-derived NPC aggregates were decellularized. The derived ECMs were crosslinked using genipin or glutaraldehyde to enhance the scaffold stability. ESC-derived NPC aggregates were reseeded on different ECM scaffolds and differential cellular compositions of neural progenitors, neurons, and glial cells were observed. The results indicate that ESC-derived ECM scaffolds affect neural differentiation through intrinsic biological cues and biophysical properties. These scaffolds have potential for in vitro cell culture and in vivo tissue regeneration study. STATEMENT OF SIGNIFICANCE Dynamic interactions of acellular extracellular matrices and stem cells are critical for lineage-specific commitment and tissue regeneration. Understanding the synergistic effects of biochemical, biological, and biophysical properties of acellular matrices would facilitate scaffold design and the functional regulation of stem cells. The present study assessed the influence of crosslinked embryonic stem cell-derived extracellular matrix on neural differentiation and revealed the synergistic interactions of various matrix properties. While embryonic stem cell-derived matrices have been assessed as tissue engineering scaffolds, the impact of crosslinking on the embryonic stem cell-derived matrices to modulate neural differentiation has not been studied. The results from this study provide novel knowledge on the interface of embryonic stem cell-derived extracellular matrix and neural aggregates. The findings reported in this manuscript are significant for stem cell differentiation toward the applications in stem cell-based drug screening, disease modeling, and cell therapies.

Neural patterning of human induced pluripotent stem cells in 3-D cultures for studying biomolecule-directed differential cellular responses

INTRODUCTION Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells/tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capacity of signaling factors that regulate 3-D neural tissue patterning in vitro and differential responses of the resulting neural populations to various biomolecules have not yet been fully understood. METHODS By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog (SHH) signaling, this study generated different 3-D neuronal cultures that were mainly comprised of either cortical glutamatergic neurons or motor neurons. RESULTS Abundant glutamatergic neurons were observed following the treatment with an antagonist of SHH signaling, cyclopamine, while Islet-1 and HB9-expressing motor neurons were enriched by an SHH agonist, purmorphamine. In neurons derived with different neural patterning factors, whole-cell patch clamp recordings showed similar voltage-gated Na(+)/K(+) currents, depolarization-evoked action potentials and spontaneous excitatory post-synaptic currents. Moreover, these different neuronal populations exhibited differential responses to three classes of biomolecules, including (1) matrix metalloproteinase inhibitors that affect extracellular matrix remodeling; (2) N-methyl-d-aspartate that induces general neurotoxicity; and (3) amyloid β (1-42) oligomers that cause neuronal subtype-specific neurotoxicity. CONCLUSIONS This study should advance our understanding of hiPSC self-organization and neural tissue development and provide a transformative approach to establish 3-D models for neurological disease modeling and drug discovery. STATEMENT OF SIGNIFICANCE Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells, tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capability of sonic hedgehog-related small molecules to tune different neuronal subtypes in 3-D differentiation from hiPSCs and the differential cellular responses of region-specific neuronal subtypes to various biomolecules have not been fully investigated. By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog signaling, this study provides knowledge on the differential susceptibility of region-specific neuronal subtypes derived from hiPSCs to different biomolecules in extracellular matrix remodeling and neurotoxicity. The findings are significant for understanding 3-D neural patterning of hiPSCs for the applications in brain organoid formation, neurological disease modeling, and drug discovery.

Intracellular labeling of mouse embryonic stem cell–derived neural progenitor aggregates with micron-sized particles of iron oxide

BACKGROUND AIMS Pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) represent an unlimited source for the treatment of various neurological disorders. NPCs are usually derived from PSCs through the formation of embryoid body (EB), an aggregate structure mimicking embryonic development. This study investigated the effect of labeling multicellular EB-NPC aggregates with micron-sized particles of iron oxide (MPIO) for cell tracking using magnetic resonance imaging (MRI). METHODS Intact and dissociated EB-NPC aggregates were labeled with various concentrations of MPIOs (0, 2.5, 5 and 10 μg Fe/mL). The labeled cells were analyzed by fluorescent imaging, flow cytometry and in vitro MRI for labeling efficiency and detectability. Moreover, the biological effects of intracellular MPIO on cell viability, cytotoxicity, proliferation and neural differentiation were evaluated. RESULTS Intact EB-NPC aggregates showed higher cell proliferation and viability compared with the dissociated cells. Despite diffusion limitation at low MPIO concentration, higher concentration of MPIO (i.e., 10 μg Fe/mL) was able to label EB-NPC aggregates at similar efficiency to the single cells. In vitro MRI showed concentration-dependent MPIO detection in EB-NPCs over 2.0-2.6 population doublings. More important, MPIO incorporation did not affect the proliferation and neural differentiation of EB-NPCs. CONCLUSIONS Multicellular EB-NPC aggregates can be efficiently labeled and tracked with MPIO while maintaining cell proliferation, phenotype and neural differentiation potential. This study demonstrated the feasibility of labeling EB-NPC aggregates with MPIO for cellular monitoring of in vitro cultures and in vivo transplantation.

Asymmetric biodegradable microdevices for cell-borne drug delivery.

Use of live cells as carriers for drug-laden particulate structures possesses unique advantages for drug delivery. In this work, we report on the development of a novel type of particulate structures called microdevices for cell-borne drug delivery. The microdevices were fabricated by soft lithography with a disklike shape. Each microdevice was composed of a layer of biodegradable thermoplastic such as poly(lactic-co-glycolic acid). One face of the thermoplastic layer was covalently grafted with a cell-adhesive polyelectrolyte such as poly-l-lysine. This asymmetric structure allowed the microdevices to bind to live cells through bulk mixing without causing cell aggregation. Moreover, the cell-microdevice complexes were largely stable, and the viability and proliferation ability of the cells were not affected by the microdevices over a week. In addition, sustained release of a mock drug from the microdevices was demonstrated. This type of microdevice promises to be clinically useful for sustained intravascular drug delivery.

Facile functionalization and assembly of live cells with microcontact-printed polymeric biomaterials.

The functionalization and assembly of live cells with microfabricated polymeric biomaterials have attracted considerable interest in recent years, but the conventional methods suffer from high cost, high complexity, long processing time or inadequate capability. The present study reports on the development of a novel method for functionalizing and assembling live cells by integrating microcontact printing of polymeric biomaterials with a temperature-sensitive sacrificial layer prepared by spin-coating. This method has been used not only to functionalize live cells with microscopic polyelectrolyte and thermoplastic structures of various sizes and shapes, but also to assemble the cells into macroscopic stripes and sheets. The method is applicable to multiple types of cells, including human leukemic cells, mouse embryonic stem cells and human mesenchymal stem cells in the forms of single cells and cell aggregates. In addition, the microcontact-printed structures can be prepared using biodegradable and biocompatible polyelectrolytes and thermoplastic. The unique combination of low cost, ease of use and high versatility renders this method potentially useful for diverse biomedical applications, including drug delivery, cell tracking and tissue engineering.

Pluripotent stem cell expansion and neural differentiation in 3-D scaffolds of tunable Poisson's ratio.

Biophysical properties of the scaffolds such as the elastic modulus, have been recently shown to impact stem cell lineage commitment. On the other hand, the contribution of the Poissons ratio, another important biophysical property, to the stem cell fate decision, has not been studied. Scaffolds with tunable Poissons ratio (ν) (termed as auxetic scaffolds when Poissons ratio is zero or negative) are anticipated to provide a spectrum of unique biophysical 3-D microenvironments to influence stem cell fate. To test this hypothesis, in the present work we fabricated auxetic polyurethane scaffolds (ν=0 to -0.45) and evaluated their effects on neural differentiation of mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells (hiPSCs). Compared to the regular scaffolds (ν=+0.30) before auxetic conversion, the auxetic scaffolds supported smaller aggregate formation and higher expression of β-tubulin III upon neural differentiation. The influences of pore structure, Poissons ratio, and elastic modulus on neural lineage commitment were further evaluated using a series of auxetic scaffolds. The results indicate that Poissons ratio may confound the effects of elastic modulus, and auxetic scaffolds with proper pore structure and Poissons ratio enhance neural differentiation. This study demonstrates that tuning the Poissons ratio of the scaffolds together with elastic modulus and microstructure would enhance the capability to generate broader, more diversified ranges of biophysical 3-D microenvironments for the modulation of cellular differentiation. STATEMENT OF SIGNIFICANCE Biophysical signaling from the substrates and scaffolds plays a critical role in neural lineage commitment of pluripotent stem cells. While the contribution of elastic modulus has been well studied, the influence of Poissons ratio along with microstructure of the scaffolds remains unknown largely due to the lack of technology to produce materials with tailorable Poissons ratio. This study fabricated auxetic polyurethane scaffolds with different elastic modulus, Poissons ratio and microstructure and evaluated neural differentiation of pluripotent stem cells. The findings add a novel angle to understand the impact of biophysical microenvironment on stem cell fate decisions.

Cryopreservation of embryonic stem cell-derived multicellular neural aggregates labeled with micron-sized particles of iron oxide for magnetic resonance imaging.

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)‐derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre‐labeled neural cells, especially in three‐dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC‐derived multicellular NPC aggregates labeled with micron‐sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70–80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post‐cryopreservation. MRI analysis showed comparable detectability for the MPIO‐labeled cells before and after cryopreservation indicated by T2 and T2* relaxation rates. Cryopreserving MPIO‐labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation.

Differentiation of Neural Progenitor Cells from Pluripotent Stem Cells in Artificial Niches

Recently pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have emerged as promising cell sources for regenerative medicine and drug discovery towards the treatments of various incurable neural diseases or injuries [1-4]. In many cases, the pathological trauma or disorder of adult central nerve system (CNS) is unable to be healed and repaired [5-7]. For instance, when spinal cord is injured, the motor neurons may lose function permanently, due to the inability of the spinal myelin to regenerate [5]. This paralysis leads to the loss of sensation below the site of spinal injury, which causes paraplegia or quadriplegia of patients [6]. Neural progenitor cells (NPCs) derived from PSCs possess the great potential to develop restorative treatments for neurodegenerative diseases, such as stroke, traumatic brain injury, spinal cord injury, or other CNS disorders [8-10]. PSC-derived NPCs can be differentiated into primary CNS lineages including neurons, astrocytes, or oligodendrocytes. As progenitors, these cells can also be cultivated for many passages without losing the differentiation capacity in vitro and easily be expanded in scalable process using bioreactors [11,12].