Liqing Song

Controlling Redox Status for Stem Cell Survival, Expansion, and Differentiation.

Reactive oxygen species (ROS) have long been considered as pathological agents inducing apoptosis under adverse culture conditions. However, recent findings have challenged this dogma and physiological levels of ROS are now considered as secondary messengers, mediating numerous cellular functions in stem cells. Stem cells represent important tools for tissue engineering, drug screening, and disease modeling. However, the safe use of stem cells for clinical applications still requires culture improvements to obtain functional cells. With the examples of mesenchymal stem cells (MSCs) and pluripotent stem cells (PSCs), this review investigates the roles of ROS in the maintenance of self-renewal, proliferation, and differentiation of stem cells. In addition, this work highlights that the tight control of stem cell microenvironment, including cell organization, and metabolic and mechanical environments, may be an effective approach to regulate endogenous ROS generation. Taken together, this paper indicates the need for better quantification of ROS towards the accurate control of stem cell fate.

Nanotopography promoted neuronal differentiation of human induced pluripotent stem cells.

Inefficient neural differentiation of human induced pluripotent stem cells (hiPSCs) motivates recent investigation of the influence of biophysical characteristics of cellular microenvironment, in particular nanotopography, on hiPSC fate decision. However, the roles of geometry and dimensions of nanotopography in neural lineage commitment of hiPSCs have not been well understood. The objective of this study is to delineate the effects of geometry, feature size and height of nanotopography on neuronal differentiation of hiPSCs. HiPSCs were seeded on equally spaced nanogratings (500 and 1000nm in linewidth) and hexagonally arranged nanopillars (500nm in diameter), each having a height of 150 or 560nm, and induced for neuronal differentiation in concert with dual Smad inhibitors. The gratings of 560nm height reduced cell proliferation, enhanced cytoplasmic localization of Yes-associated protein, and promoted neuronal differentiation (up to 60% βIII-tubulin+ cells) compared with the flat control. Nanograting-induced cell polarity and cytoplasmic YAP localization were shown to be critical to the induced neural differentiation of hiPSCs. The derived neuronal cells express MAP2, Tau, glutamate, GABA and Islet-1, indicating the existence of multiple neuronal subtypes. This study contributes to the delineation of cell-nanotopography interactions and provides the insights into the design of nanotopography configuration for pluripotent stem cell neural lineage commitment.

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.

Wnt-YAP interactions in the neural fate of human pluripotent stem cells and the implications for neural organoid formation

ABSTRACT Human pluripotent stem cells (hPSCs) have shown the ability to self-organize into different types of neural organoids (e.g., whole brain organoids, cortical spheroids, midbrain organoids etc.) recently. The extrinsic and intrinsic signaling elicited by Wnt pathway, Hippo/Yes-associated protein (YAP) pathway, and extracellular microenvironment plays a critical role in brain tissue morphogenesis. This article highlights recent advances in neural tissue patterning from hPSCs, in particular the role of Wnt pathway and YAP activity in this process. Understanding the Wnt-YAP interactions should provide us the guidance to predict and modulate brain-like tissue structure through the regulation of extracellular microenvironment of hPSCs.

Wnt-YAP Interactions during Neural Tissue Patterning of Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) have special ability to self-assemble into neural spheroids or mini-brain-like structures. During the self-assembly process, Wnt signaling plays an important role in regional patterning and establishing positional identity of hiPSC-derived neural progenitors. Recently, the role of Wnt signaling in regulating Yes-associated protein (YAP) expression (nuclear or cytoplasmic), the pivotal regulator during organ growth and tissue generation, has attracted increasing interests. However, the interactions between Wnt and YAP expression for neural lineage commitment of hiPSCs remain poorly explored. The objective of this study is to investigate the effects of Wnt signaling and YAP expression on the cellular population in three-dimensional (3D) neural spheroids derived from hiPSCs. In this study, Wnt signaling was activated using CHIR99021 for 3D neural spheroids derived from human iPSK3 cells through embryoid body formation. Our results indicate that Wnt activation induces nuclear localization of YAP and upregulates the expression of HOXB4, the marker for hindbrain/spinal cord. By contrast, the cells exhibit more rostral forebrain neural identity (expression of TBR1) without Wnt activation. Cytochalasin D was then used to induce cytoplasmic YAP and the results showed the decreased HOXB4 expression. In addition, the incorporation of microparticles in the neural spheroids was investigated for the perturbation of neural patterning. This study may indicate the bidirectional interactions of Wnt signaling and YAP expression during neural tissue patterning, which have the significance in neurological disease modeling, drug screening, and neural tissue regeneration.

Vascular differentiation from pluripotent stem cells in 3-D auxetic scaffolds

Auxetic scaffolds, that is, scaffolds that can display negative Poissons ratio, have unique physical properties and can expand transversally when axially strained or contract under compression. Auxetic materials have been used for bioprostheses and artery stents due to the enhanced compressive strength and shear stiffness. In vascular tissue engineering, auxetic scaffolds allow the widening of blood vessels when blood flows through (creating compressive stress) to prevent the blockage. However, the influence of auxetic materials on the cellular fate decision in local environment is unclear. In this study, auxetic polyurethane foams were used to support vascular differentiation from pluripotent stem cells. The expression of alkaline phosphatase, Oct‐4, and Nanog was lower after 4 days of differentiation for the cells grown in auxetic scaffolds. Higher expression of vascular markers CD31 and VE‐cadherin was observed for the cells from auxetic scaffolds compared with those from the scaffolds before auxetic conversion. Little influence on the expression of cardiac marker α‐actinin was observed. The vascular cells secreted extracellular matrix proteins vitronectin and laminin and expressed membrane‐bound matrix metalloproteinase 9. The examination of Yes‐associated protein expression indicated more cytoplasmic retention in the cells from auxetic scaffolds compared with those from regular scaffolds, suggesting that the auxetic scaffolds may affect cellular contraction. This study demonstrates a novel 3‐D culture based on auxetic scaffolds for vascular differentiation and provides a platform to study the influence of biophysical microenvironments on differentiation of pluripotent stem cells. The outcome of this study has implications for regenerative medicine and drug discovery.

Neuroprotective Activities of Heparin, Heparinase III, and Hyaluronic Acid on the Aβ42-Treated Forebrain Spheroids Derived from Human Stem Cells

Extracellular matrix (ECM) components of the brain play complex roles in neurodegenerative diseases. The study of microenvironment of brain tissues with Alzheimers disease revealed colocalized expression of different ECM molecules such as heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), matrix metal-loproteinases (MMPs), and hyaluronic acid. In this study, both cortical and hippocampal populations were generated from human-induced pluripotent stem cell-derived neural spheroids. The cultures were then treated with heparin (competes for Aβ affinity with HSPG), heparinase III (digests HSPGs), chondroitinase (digests CSPGs), hyaluronic acid, and an MMP-2/9 inhibitor (SB-3CT) together with amyloid β (Aβ42) oligomers. The results indicate that inhibition of HSPG binding to Aβ42 using either heparinase III or heparin reduces Aβ42 expression and increases the population of β-tubulin III+ neurons, whereas the inhibition of MMP2/9 induces more neurotoxicity. The results should enhance our understanding of the contribution of ECMs to the Aβ-related neural cell death.

Neural Differentiation of Spheroids Derived from HiPSC-MSC Co-Culture

Organoids, the condensed three-dimensional (3D) tissues emerged at the early stage of organogenesis, are a promising approach to regenerate functional and vascularized organ mimics. While incorporation of heterotypic cell types, such as human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cells (hiPSCs)-derived neural progenitors aid neural organ development, the interactions of secreted factors during neurogenesis have not been well understood. The objective of this study is to investigate the impact of the composition and structure of 3D hybrid spheroids of hiPSCs and hMSCs on dorsal cortical differentiation and the secretion of extracellular matrices and trophic factors in vitro. The hybrid spheroids were formed at different hiPSC:hMSC ratios (100:0, 75:25, 50:50, 25:75, 0:100) using direct mixing or pre-hiPSC aggregation method, which generated dynamic spheroid structure. The cellular organization, proliferation, neural marker expression, and the secretion of extracellular matrix proteins and the cytokines were characterized. The incorporation of MSCs upregulated Nestin and β-tubulin III expression (the dorsal cortical identity was shown by Pax6 and TBR1 expression), matrix remodeling proteins, and the secretion of transforming growth factor-β1 and prostaglandin E2. This study indicates that the appropriate composition and structure of hiPSC-MSC spheroids promote neural differentiation and trophic factor and matrix secretion due to the heterotypic cell-cell interactions.

Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor

Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.