Project description:Human pluripotent stem cells (hPSCs) offer considerable potential for biomedical applications including drug screening and cell replacement therapies. Clinical translation of hPSCs requires large quantities of high quality cells, so scalable methods for cell culture are needed. However, current methods are limited by scalability, the use of animal-derived components, and/or low expansion rates. A thermoresponsive 3D hydrogel for scalable hPSC expansion and differentiation into several defined lineages is recently reported. This system would benefit from increased control over material properties to further tune hPSC behavior, and here a scalable 3D biomaterial with the capacity to tune both the chemical and the mechanical properties is demonstrated to promote hPSC expansion under defined conditions. This 3D biomaterial, comprised of hyaluronic acid and poly(N-isopropolyacrylamide), has thermoresponsive properties that readily enable mixing with cells at low temperatures, physical encapsulation within the hydrogel upon elevation at 37 °C, and cell recovery upon cooling and reliquefaction. After optimization, the resulting biomaterial supports hPSC expansion over long cell culture periods while maintaining cell pluripotency. The capacity to modulate the mechanical and chemical properties of the hydrogel provides a new avenue to expand hPSCs for future therapeutic application.
Project description:This study was designed to investigate the regulatory effect of hyaluronic acid (HA)-coating silk fibroin (SF) nanofibers during epithelialization of urinary tract for urethral regeneration. The obtained electrospun biomimetic tubular HA-SF nanofiber scaffold is composed of a dense inner layer and a porous outer layer in order to mimic adhesion and cavernous layers of the native tissue, respectively. A thin layer of HA-gel coating was fixed in the inner wall to provide SF nanofibers with a dense and smooth surface nano-topography and higher hydrophilicity. Compared with pure SF nanofibers, HA-SF nanofibers significantly promoted the adhesion, growth, and proliferation of primary urothelial cells, and up-regulate the expression of uroplakin-3 (terminal differentiation keratin protein in urothelium). Using the New Zealand male rabbit urethral injury model, the scaffold composed of tubular HA-SF nanofibers could recruit lumen and myoepithelial cells from the adjacent area of the host, rapidly reconstructing the urothelial barrier in the wound area in order to keep the urinary tract unobstructed, thereby promoting luminal epithelialization, smooth muscle bundle structural remodeling, and capillary formation. Overall, the synergistic effects of nano-topography and biophysical cues in a biomimetic scaffold design for effective endogenous regeneration.
Project description:Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from a three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from a monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stem medium (MiPS) adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR-derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were upregulated in spheroids at day 6 and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were replated in normal FBS medium, cells formed a typical spindle-shaped morphology and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.
Project description:3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30-500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(ɛ-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca2+ imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases.
Project description:The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments.
Project description:Repair or regeneration of damaged nerves is still a challenging clinical task in reconstructive surgeries and regenerative medicine. Here, it is demonstrated that periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) isolated from adult human periodontal and gingival tissues assume neuronal phenotype in vitro and in vivo via a subcutaneous transplantation model in nude mice. PDLSCs and GMSCs are encapsulated in a 3D scaffold based on alginate and hyaluronic acid hydrogels capable of sustained release of human nerve growth factor (NGF). The elasticity of the hydrogels affects the proliferation and differentiation of encapsulated MSCs within scaffolds. Moreover, it is observed that PDLSCs and GMSCs are stained positive for βIII-tubulin, while exhibiting high levels of gene expression related to neurogenic differentiation (βIII-tubulin and glial fibrillary acidic protein) via quantitative polymerase chain reaction (qPCR). Western blot analysis shows the importance of elasticity of the matrix and the presence of NGF in the neurogenic differentiation of encapsulated MSCs. In vivo, immunofluorescence staining for neurogenic specific protein markers confirms islands of dense positively stained structures inside transplanted hydrogels. As far as it is known, this study is the first demonstration of the application of PDLSCs and GMSCs as promising cell therapy candidates for nerve regeneration.
Project description:Hematopoietic stem progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic "niche," a special 3-dimensional (3D) microenvironment that regulates HSPC self-renewal and multipotency. In this study, we evaluated a novel 3D in vitro culture system that uses components of the BM hematopoietic niche to expand umbilical cord blood (UCB) CD34+ cells. We developed this model using decellularized Wharton jelly matrix (DWJM) as an extracellular matrix (ECM) scaffold and human BM mesenchymal stromal cells (MSCs) as supporting niche cells. To assess the efficacy of this model in expanding CD34+ cells, we analyzed UCB CD34+ cells, following culture in DWJM, for proliferation, viability, self-renewal, multilineage differentiation, and transmigration capability. We found that DWJM significantly expanded UCB HSPC subset. It promoted UCB CD34+ cell quiescence, while maintaining their viability, differentiation potential with megakaryocytic differentiation bias, and clonogenic capacity. DWJM induced an increase in the frequency of c-kit+ cells, a population with enhanced self-renewal ability, and in CXCR4 expression in CD34+ cells, which enhanced their transmigration capability. The presence of BM MSCs in DWJM, however, impaired UCB CD34+ cell transmigration and suppressed CXCR4 expression. Transcriptome analysis indicated that DWJM upregulates a set of genes that are specifically involved in megakaryocytic differentiation, cell mobility, and BM homing. Collectively, our results indicate that the DWJM-based 3D culture system is a novel in vitro model that supports the proliferation of UCB CD34+ cells with enhanced transmigration potential, while maintaining their differentiation potential. Our findings shed light on the interplay between DWJM and BM MSCs in supporting the ex vivo culture of human UCB CD34+ cells for use in clinical transplantation.
Project description:Bone fractures and defects pose serious health-related issues on patients. For clinical therapeutics, synthetic scaffolds have been actively explored to promote critical-sized bone regeneration, and electrical stimulations are recognized as an effective auxiliary to facilitate the process. Here, we develop a three-dimensional (3D) biomimetic scaffold integrated with thin-film silicon (Si)-based microstructures. This Si-based hybrid scaffold not only provides a 3D hierarchical structure for guiding cell growth but also regulates cell behaviors via photo-induced electrical signals. Remotely controlled by infrared illumination, these Si structures electrically modulate membrane potentials and intracellular calcium dynamics of stem cells and potentiate cell proliferation and differentiation. In a rodent model, the Si-integrated scaffold demonstrates improved osteogenesis under optical stimulations. Such a wirelessly powered optoelectronic scaffold eliminates tethered electrical implants and fully degrades in a biological environment. The Si-based 3D scaffold combines topographical and optoelectronic stimuli for effective biological modulations, offering broad potential for biomedicine.
Project description:The self-organizing properties of stem cells have been exploited to generate organoids, organ-specific, cell-containing, three-dimensional (3D) structures. The present study aimed to introduce a novel bioengineering technique for driving the effective organization of adult tissue stem cells via niche-independent 3D microwell culture. Microwells were fabricated by photopatterning poly(ethylene glycol) hydrogel in the presence of an electrospun polycaprolactone nanofibrous scaffold. Human single clonal salivary gland stem cells (SGSCs) were cultured in nanofibrous microwells through two simple steps, priming and differentiation. Before the induction of 3D organization, single clonal SGSCs were preconditioned to aggregate to form 3D spheroids in different matrices, such as Matrigel, floating dish, and microwells. Expression of salivary stem cell markers and pluripotency markers was greater in 3D spheroid cultures than in 2D plastic culture. Lobular structures were organized by changing media, and those in microwells exhibited higher salivary acinar, ductal, and tight junction marker levels and decreased stem-cell marker levels relative to other 3D cultures. Furthermore, higher α-amylase secretion and intracellular calcium levels were observed in the presence of adrenergic or cholinergic agonists, respectively, along with an increased intracellular pH, suggesting more functional salivary organoid formation. These microwell-driven organoids also engrafted successfully into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Our results showed that microwell-cultured SGSCs organize into salivary structures and that this biomimetic 3D culture technique can promote effective generation of niche-independent single stem-cell-based 3D organoids.