Microfluidic Investigation of BDNF‐Enhanced Neural Stem Cell Chemotaxis in CXCL12 Gradients

In vivo studies have suggested that gradients of CXCL12 (aka stromal cell‐derived factor 1α) may be critical for neural stem cell (NSC) migration during brain development and neural tissue regeneration. However, traditional in vitro chemotaxis tools are limited by unstable concentration gradients and the inability to decouple cell migration directionality and speed. These limitations have restricted the reproducible and quantitative analysis of neuronal migration, which is required for mechanism‐based studies. Using a microfluidic gradient generator, nestin and Sox‐2 positive human embryonic NSC chemotaxis is quantified within a linear and stable CXCL12 gradient. While untreated NSCs are not able to chemotax within CXCL12 gradients, pre‐treatment of the cells with brain‐derived neurotrophic factor (BDNF) results in significant chemotactic, directional migration. BDNF pre‐treatment has no effect on cell migration speed, which averages about 1 μm min^?1. Quantitative analysis determines that CXCL12 concentrations above 9.0 nM are above the minimum activation threshold, while concentrations below 14.7 nM are below the saturation threshold. Interestingly, although inhibitor studies with AMD 3100 revealed that CXCL12 chemotaxis requires receptor CXCR4 activation, BDNF pre‐treatment is found to have no profound effects on the mRNA levels or surface presentation of CXCR4 or the putative CXCR7 scavenger receptor. The microfluidic study of NSC migration within stable chemokine concentration profiles provides quantitative analysis as well as new insight into the migratory mechanism underlying BDNF‐induced chemotaxis towards CXCL12.     

Cellular Stemness Maintenance of Human Adipose‐Derived Stem Cells on ZnO Nanorod Arrays

Ever‐growing tissue regeneration and other stem cell therapies cause pressing need for large population of self‐renewable stem cells. However, stem cells gradually lose their stemness after long‐term in vitro cultivation. In this study, a ZnO nanorod (ZnO NR) array is used to maintain the stemness of human adipose‐derived stem cells (hADSCs). The results prove that after culturing hADSCs on ZnO NRs for 3 weeks, the stemness genes and protein expression level are higher than that on culture plates and ZnO film. ZnO NRs can maintain stemness of hADSCs without inhibiting the cell proliferation and oriented differentiation capabilities. KLF4 (Kruppel‐like factor 4) is a Zn²⁺‐binding gene that plays a vital role in cell proliferation and differentiation. Sustained Zn²⁺ release and the increased expression of KLF4 can be detected, suggesting that ZnO NRs have efficiently released Zn²⁺ for stemness maintenance. Taken together, the nanotopography of ZnO NRs and the Zn²⁺ release synergistically facilitate stemness maintenance. This study has provided a powerful tool for directing cell fate, maintaining stemness, and realizing the expansion of stem cells in vitro, which will open a new route for the manufacture of large populations of stem cells and fulfilling the growing demand for the cell therapy market.     

Self‐Assembling Proteins as High‐Performance Substrates for Embryonic Stem Cell Self‐Renewal

The development of extracellular matrix mimetics that imitate niche stem cell microenvironments and support cell growth for technological applications is intensely pursued. Specifically, mimetics are sought that can enact control over the self‐renewal and directed differentiation of human pluripotent stem cells (hPSCs) for clinical use. Despite considerable progress in the field, a major impediment to the clinical translation of hPSCs is the difficulty and high cost of large‐scale cell production under xeno‐free culture conditions using current matrices. Here, a bioactive, recombinant, protein‐based polymer, termed ZT^Fn, is presented that closely mimics human plasma fibronectin and serves as an economical, xeno‐free, biodegradable, and functionally adaptable cell substrate. The ZT^Fn substrate supports with high performance the propagation and long‐term self‐renewal of human embryonic stem cells while preserving their pluripotency. The ZT^Fn polymer can, therefore, be proposed as an efficient and affordable replacement for fibronectin in clinical grade cell culturing. Further, it can be postulated that the ZT polymer has significant engineering potential for further orthogonal functionalization in complex cell applications.     

Low Forces Push the Maturation of Neural Precursors into Neurons

Mechanical stimulation modulates neural development and neuronal activity. In a previous study, magnetic “nano-pulling” is proposed as a tool to generate active forces. By loading neural cells with magnetic nanoparticles (MNPs), a precise force vector is remotely generated through static magnetic fields. In the present study, human neural stem cells (NSCs) are subjected to a standard differentiation protocol, in the presence or absence of nano-pulling. Under mechanical stimulation, an increase in the length of the neural processes which showed an enrichment in microtubules, endoplasmic reticulum, and mitochondria is found. A stimulation lasting up to 82 days induces a strong remodeling at the level of synapse density and a re-organization of the neuronal network, halving the time required for the maturation of neural precursors into neurons. The MNP-loaded NSCs are then transplanted into mouse spinal cord organotypic slices, demonstrating that nano-pulling stimulates the elongation of the NSC processes and modulates their orientation even in an ex vivo model. Thus, it is shown that active mechanical stimuli can guide the outgrowth of NSCs transplanted into the spinal cord tissue. The findings suggest that mechanical forces play an important role in neuronal maturation which could be applied in regenerative medicine.     

“All‐in‐One” Gel System for Whole Procedure of Stem‐Cell Amplification and Tissue Engineering

Microsphere (MS)‐based systems provides great advantages for cell expansion and transplantation due to their high surface‐to‐volume ratio and biomimetic environment. However, a MS‐based system that includes cell attachment, proliferation, passage, harvest, cryopreservation, and tissue engineering together has not been realized yet. An “all‐in‐one” gel MS‐based system is established for human adipose‐derived mesenchymal stem cells (hADSCs), realizing real 3D culture with enhanced expansion efficiency and simplified serial cell culture operations, and construction of macrotissues with uniform cell distribution and specific function. A 3D digital light‐processing technology is developed to fabricate gel MSs in an effective way. The printed MSs present a suitable environment with rough surface architecture and the mechanical properties of soft tissues, leading to high cell viability, attachment, proliferation, activity, and differentiation potential. Further, convenient standard operation procedures, including cell passage, detachment, and cryopreservation, are established for cell culture on the gel MSs. Finally, hADSCs‐loaded gel MSs form macrotissues through a “bottom‐up” approach, which demonstrates the potential applications for tissue engineering. These findings exhibit the feasibility and beauty of “all‐in‐one” stem cell culture and tissue engineering system.     

Effective Gene Delivery into Human Stem Cells with a Cell‐Targeting Peptide‐Modified Bioreducible Polymer

Stem cells are poorly permissive to non‐viral gene transfection reagents. In this study, we explored the possibility of improving gene delivery into human embryonic (hESC) and mesenchymal (hMSC) stem cells by synergizing the activity of a cell‐binding ligand with a polymer that releases nucleic acids in a cytoplasm‐responsive manner. A 29 amino acid long peptide, RVG, targeting the nicotinic acetylcholine receptor (nAchR) was identified to bind both hMSC and H9‐derived hESC. Conjugating RVG to a redox‐sensitive biodegradable dendrimer‐type arginine‐grafted polymer (PAM‐ABP) enabled nanoparticle formation with plasmid DNA without altering the environment‐sensitive DNA release property and favorable toxicity profile of the parent polymer. Importantly, RVG‐PAM‐ABP quantitatively enhanced transfection into both hMSC and hESC compared to commercial transfection reagents like Lipofectamine 2000 and Fugene. ～60% and 50% of hMSC and hESC were respectively transfected, and at increased levels on a per cell basis, without affecting pluripotency marker expression. RVG‐PAM‐ABP is thus a novel bioreducible, biocompatible, non‐toxic, synthetic gene delivery system for nAchR‐expressing stem cells. Our data also demonstrates that a cell‐binding ligand like RVG can cooperate with a gene delivery system like PAM‐ABP to enable transfection of poorly‐permissive cells.     

ZnO/Nanocarbons‐Modified Fibrous Scaffolds for Stem Cell‐Based Osteogenic Differentiation

Currently, mesenchymal stem cells (MSCs)‐based therapies for bone regeneration and treatments have gained significant attention in clinical research. Though many chemical and physical cues which influence the osteogenic differentiation of MSCs have been explored, scaffolds combining the benefits of Zn²⁺ ions and unique nanostructures may become an ideal interface to enhance osteogenic and anti‐infective capabilities simultaneously. In this work, motivated by the enormous advantages of Zn‐based metal–organic framework‐derived nanocarbons, C‐ZnO nanocarbons‐modified fibrous scaffolds for stem cell‐based osteogenic differentiation are constructed. The modified scaffolds show enhanced expression of alkaline phosphatase, bone sialoprotein, vinculin, and a larger cell spreading area. Meanwhile, the caging of ZnO nanoparticles can allow the slow release of Zn²⁺ ions, which not only activate various signaling pathways to guide osteogenic differentiation but also prevent the potential bacterial infection of implantable scaffolds. Overall, this study may provide new insight for designing stem cell‐based nanostructured fibrous scaffolds with simultaneously enhanced osteogenic and anti‐infective capabilities.     

Mussel Adhesion‐Inspired Reverse Transfection Platform Enhances Osteogenic Differentiation and Bone Formation of Human Adipose‐Derived Stem Cells

Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution‐mediated transfection are limited due to low transfection efficiency and insufficient duration of cell‐siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio‐inspired polymer‐mediated reverse transfection system is developed consisting of implantable poly(lactic‐co‐glycolic acid) (PLGA) scaffolds functionalized with siRNA‐lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating. Immobilized sLNP complexes are stably maintained without any loss of siRNA on the pDA‐coated scaffolds for 2 weeks, likely due to the formation of strong covalent bonds between amine groups of sLNP and catechol group of pDA. siRNA reverse transfection with the pDA‐sLNP‐PLGA system does not exhibit cytotoxicity and induces efficient silencing of an osteogenesis inhibitor gene in human adipose‐derived stem cells (hADSCs), resulting in enhanced osteogenic differentiation of hADSCs. Finally, hADSCs osteogenically committed on the pDA‐sLNP‐PLGA scaffolds enhanced bone formation in a mouse model of critical‐sized bone defect. Therefore, the bio‐inspired reverse transfection system can provide an all‐in‐one platform for genetic modification, differentiation, and transplantation of stem cells, simultaneously enabling both stem cell manipulation and tissue engineering.     

Nondestructive Real‐Time Monitoring of Enhanced Stem Cell Differentiation Using a Graphene‐Au Hybrid Nanoelectrode Array

Stem cells have attracted increasing research interest in the field of regenerative medicine because of their unique ability to differentiate into multiple cell lineages. However, controlling stem cell differentiation efficiently and improving the current destructive characterization methods for monitoring stem cell differentiation are the critical issues. To this end, multifunctional graphene–gold (Au) hybrid nanoelectrode arrays (NEAs) to: (i) investigate the effects of combinatorial physicochemical cues on stem cell differentiation, (ii) enhance stem cell differentiation efficiency through biophysical cues, and (iii) characterize stem cell differentiation in a nondestructive real‐time manner are developed. Through the synergistic effects of physiochemical properties of graphene and biophysical cues from nanoarrays, the graphene‐Au hybrid NEAs facilitate highly enhanced cell adhesion and spreading behaviors. In addition, by varying the dimensions of the graphene‐Au hybrid NEAs, improved stem cell differentiation efficiency, resulting from the increased focal adhesion signal, is shown. Furthermore, graphene‐Au hybrid NEAs are utilized to monitor osteogenic differentiation of stem cells electrochemically in a nondestructive real‐time manner. Collectively, it is believed the unique multifunctional graphene‐Au hybrid NEAs can significantly advance stem‐cell‐based biomedical applications.     

Injectable and Crosslinkable PLGA‐Based Microribbons as 3D Macroporous Stem Cell Niche

Poly(lactide‐co‐glycolide) (PLGA) has been widely used as a tissue engineering scaffold. However, conventional PLGA scaffolds are not injectable, and do not support direct cell encapsulation, leading to poor cell distribution in 3D. Here, a method for fabricating injectable and intercrosslinkable PLGA microribbon‐based macroporous scaffolds as 3D stem cell niche is reported. PLGA is first fabricated into microribbon‐shape building blocks with tunable width using microcontact printing, then coated with fibrinogen to enhance solubility and injectability using aqueous solution. Upon mixing with thrombin, firbornogen‐coated PLGA microribbons can intercrosslink into 3D scaffolds. When subject to cyclic compression, PLGA microribbon scaffolds exhibit great shock‐absorbing capacity and return to their original shape, while conventional PLGA scaffolds exhibit permanent deformation after one cycle. Using human mesenchymal stem cells (hMSCs) as a model cell type, it is demonstrated that PLGA μRB scaffolds support homogeneous cell encapsulation, and robust cell spreading and proliferation in 3D. After 28 days of culture in osteogenic medium, hMSC‐seeded PLGA μRB scaffolds exhibit an increase in compressive modulus and robust bone formation as shown by staining of alkaline phosphatase, mineralization, and collagen. Together, the results validate PLGA μRBs as a promising injectable, macroporous, non‐hydrogel‐based scaffold for cell delivery and tissue regeneration applications.     

A Micro‐Ark for Cells: Highly Open Porous Polyhydroxyalkanoate Microspheres as Injectable Scaffolds for Tissue Regeneration

To avoid large open surgery using scaffold transplants, small‐sized cell carriers are employed to repair complexly shaped tissue defects. However, most cell carriers show poor cell adherences and viability. Therefore, polyhydroxyalkanoate (PHA), a natural biopolymer, is used to prepare highly open porous microspheres (OPMs) of 300–360 µm in diameter, combining the advantages of microspheres and scaffolds to serve as injectable carriers harboring proliferating stem cells. In addition to the convenient injection to a defected tissue, and in contrast to poor performances of OPMs made of polylactides (PLA OPMs) and traditional less porous hollow microspheres (PHA HMs), PHA OPMs present suitable surface pores of 10–60 µm and interconnected passages with an average size of 8.8 µm, leading to a high in vitro cell adhesion of 93.4%, continuous proliferation for 10 d and improved differentiation of human bone marrow mesenchymal stem cells (hMSCs). PHA OPMs also support stronger osteoblast‐regeneration compared with traditional PHA HMs, PLA OPMs, commercial hyaluronic acid hydrogels, and carrier‐free hMSCs in an ectopic bone‐formation mouse model. PHA OPMs protect cells against stresses during injection, allowing more living cells to proliferate and migrate to damaged tissues. They function like a micro‐Noah's Ark to safely transport cells to a defect tissue.     

Carbon nanotube rope with electrical stimulation promotes the differentiation and maturity of neural stem cells

In recent years, the utilization of nanomaterials such as carbon nanotubes (CNTs) in the field of neuroscience has forever changed the approach to nerve-related research. The array of novel properties CNTs possess allows them to interact with neurons at the nanodimensional scale. In this study, a CNT rope substrate is developed to allow the electrical stimulation of neural stem cells (NSCs) in culture medium and the in situ observation of the response of these stem cells after stimulation. CNTs are synthesized by chemical vapor deposition and prepared into a ropelike structure with a diameter of 1 mm and length of 1.5 cm. NSCs are differentiated on the CNT rope substrate while the direction of neurite outgrowth, phenotype, and maturity of the NSCs are analyzed. Fluorescence and scanning electron microscopy demonstrate that neurite extension favors the direction of the spiral topography on the CNT rope. NSCs plated on CNT ropes are boosted towards differentiated neurons in the early culture stage when compared to conventional tissue culture plates via the analysis of neuronal gene and protein expressions by quantitative polymerase chain reaction and immunostaining, respectively. Furthermore, a set of electrical stimulation parameters (5 mV, 0.5 mA, 25 ms intermittent stimulation) promotes neuronal maturity while also increasing the speed of neurite outgrowth. These results indicate that an electroconductive CNT rope substrate along with electrical stimulation may have a synergistic effect on promoting neurite elongation and boosting effects on the differentiation of NSCs into mature neuronal cells for therapeutic application in neural regeneration.     

Revealing the Fate of Transplanted Stem Cells In Vivo with a Novel Optical Imaging Strategy

Stem‐cell‐based regenerative medicine holds great promise in clinical practices. However, the fate of stem cells after transplantation, including the distribution, viability, and the cell clearance, is not fully understood, which is critical to understand the process and the underlying mechanism of regeneration for better therapeutic effects. Herein, we develop a dual‐labeling strategy to in situ visualize the fate of transplanted stem cells in vivo by combining the exogenous near‐infrared fluorescence imaging in the second window (NIR‐II) and endogenous red bioluminescence imaging (BLI). The NIR‐II fluorescence of Ag₂S quantum dots is employed to dynamically monitor the trafficking and distribution of all transplanted stem cells in vivo due to its deep tissue penetration and high spatiotemporal resolution, while BLI of red‐emitting firefly luciferase (RfLuc) identifies the living stem cells after transplantation in vivo because only the living stem cells express RfLuc. This facile strategy allows for in situ visualization of the dynamic trafficking of stem cells in vivo and the quantitative evaluation of cell translocation and viability with high temporal and spatial resolution, and thus reports the fate of transplanted stem cells and how the living stem cells help, regeneration, for an instance, of a mouse with acute liver failure.     

Magnetically Controlled Growth‐Factor‐Immobilized Multilayer Cell Sheets for Complex Tissue Regeneration

The scaffold‐free cell‐sheet technique plays a significant role in stem‐cell‐based regeneration. Furthermore, growth factors are known to direct stem cell differentiation and enhance tissue regeneration. However, the absence of an effective means to incorporate growth factors into the cell sheets hinders further optimization of the regeneration efficiency. Here, a novel design of magnetically controlled “growth‐factor‐immobilized cell sheets” is reported. A new Fe₃O₄ magnetic nanoparticle (MNP) coated with nanoscale graphene oxide (nGO@Fe₃O₄) is developed to label stem cells and deliver growth factors. First, the nGO@Fe₃O₄ MNPs can be easily swallowed by dental‐pulp stem cells (DPSCs) and have no influence on cell viability. Thus, the MNP‐labeled cells can be organized via magnetic force to form multilayered cell sheets in different patterns. Second, compared to traditional Fe₃O₄ nanoparticles, the graphene oxide coating provides plenty of carboxyl groups to bind and deliver growth factors. Therefore, with these nGO@Fe₃O₄ MNPs, bone‐morphogenetic‐protein‐2 (BMP2) is successfully incorporated into the DPSCs sheets to induce more bone formation. Furthermore, an integrated osteochondral complex is also constructed using a combination of DPSCs/TGFβ3 and DPSCs/BMP2. All these results demonstrate that the new cell‐sheet tissue‐engineering approach exhibits promising potential for future use in regenerative medicine.     

Biomimetic Nanosilica–Collagen Scaffolds for In Situ Bone Regeneration: Toward a Cell‐Free,One‐Step Surgery

Current approaches to fabrication of nSC composites for bone tissue engineering (BTE) have limited capacity to achieve uniform surface functionalization while replicating the complex architecture and bioactivity of native bone, compromising application of these nanocomposites for in situ bone regeneration. A robust biosilicification strategy is reported to impart a uniform and stable osteoinductive surface to porous collagen scaffolds. The resultant nSC composites possess a native‐bone‐like porous structure and a nanosilica coating. The osteoinductivity of the nSC scaffolds is strongly dependent on the surface roughness and silicon content in the silica coating. Notably, without the use of exogenous cells and growth factors (GFs), the nSC scaffolds induce successful repair of a critical‐sized calvarium defect in a rabbit model. It is revealed that topographic and chemical cues presented by nSC scaffolds could synergistically activate multiple signaling pathways related to mesenchymal stem cell recruitment and bone regeneration. Thus, this facile surface biosilicification approach could be valuable by enabling production of BTE scaffolds with large sizes, complex porous structures, and varied osteoinductivity. The nanosilica‐functionalized scaffolds can be implanted via a cell/GF‐free, one‐step surgery for in situ bone regeneration, thus demonstrating high potential for clinical translation in treatment of massive bone defects.     

Mesenchymal Stem Cell Capping on ECM‐Anchored Caspase Inhibitor–Loaded PLGA Microspheres for Intraperitoneal Injection in DSS‐Induced Murine Colitis

Mesenchymal stem cells (MSCs) are considered as a promising alternative for the treatment of various inflammatory disorders. However, poor viability and engraftment of MSCs after transplantation are major hurdles in mesenchymal stem cell therapy. Extracellular matrix (ECM)‐coated scaffolds provide better cell attachment and mechanical support for MSCs after transplantation. A single‐step method for ECM functionalization on poly(lactic‐co‐glycolic acid) (PLGA) microspheres using a novel compound, dopamine‐conjugated poly(ethylene‐alt‐maleic acid), as a stabilizer during the preparation of microspheres is reported. The dopamine molecules on the surface of microspheres provide active sites for the conjugation of ECM in an aqueous solution. The results reveal that the viability of MSCs improves when they are coated over the ECM‐functionalized PLGA microspheres (eMs). In addition, the incorporation of a broad‐spectrum caspase inhibitor (IDN6556) into the eMs synergistically increases the viability of MSCs under in vitro conditions. Intraperitoneal injection of the MSC–microsphere hybrid alleviates experimental colitis in a murine model via inhibiting Th1 and Th17 differentiation of CD4⁺ T cells in colon‐draining mesenteric lymph nodes. Therefore, drug‐loaded ECM‐coated surfaces may be considered as attractive tools for improving viability, proliferation, and functionality of MSCs following transplantation.     

Deconstructed Microfluidic Bone Marrow On‐A‐Chip to Study Normal and Malignant Hemopoietic Cell–Niche Interactions

Human hematopoietic niches are complex specialized microenvironments that maintain and regulate hematopoietic stem and progenitor cells (HSPC). Thus far, most of the studies performed investigating alterations of HSPC‐niche dynamic interactions are conducted in animal models. Herein, organ microengineering with microfluidics is combined to develop a human bone marrow (BM)‐on‐a‐chip with an integrated recirculating perfusion system that consolidates a variety of important parameters such as 3D architecture, cell–cell/cell–matrix interactions, and circulation, allowing a better mimicry of in vivo conditions. The complex BM environment is deconvoluted to 4 major distinct, but integrated, tissue‐engineered 3D niche constructs housed within a single, closed, recirculating microfluidic device system, and equipped with cell tracking technology. It is shown that this technology successfully enables the identification and quantification of preferential interactions—homing and retention—of circulating normal and malignant HSPC with distinct niches.     

Aligned Nanofiber Topographies Enhance the Differentiation of Adult Renal Stem Cells into Glomerular Podocytes

The use of adult stem/progenitor cells is a challenge in current research about kidney functional regeneration. In this framework, material‐induced stem cell differentiation can become a new paradigm to promote advances in therapies. Here, the effects of both isotropic and anisotropic fibrous topographies on the podocyte differentiation of adult renal stem cells (RSCs) from human donors are investigated. The proliferation rate of RSCs is analyzed, immunofluorescence and genetic analyses of specific podocyte markers (Wilms’ tumor 1 gene, nephrin, and podocin) are performed to assess the differentiation level on the scaffolds. The studied markers are over‐expressed in RSCs cultured on aligned fibers compared to cells cultured on either protein‐functionalized films or randomly oriented fibers. In addition, RSCs cultured on aligned fibers are found to differentiate toward podocyte precursors even in basal medium conditions, thus highlighting scaffold‐induced commitment without exogenous chemicals or cellular reprogramming. Aligned polymer fiber scaffolds which provide instructive cues for RSC differentiation might lead to new biomimetic systems for renal stem cell engineering.