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We have designed and validated a set of robust and non-toxic protocols for directly evaluating the properties of engineered neural tissue. These protocols characterize the mechanical properties of engineered neural tissues and measure their electrophysical activity. The protocols obtain elastic moduli of very soft fibrin hydrogel scaffolds and voltage readings from motor neuron cultures. Neurons require soft substrates to differentiate and mature,however measuring the elastic moduli of soft substrates remains difficult to accurately measure using standard protocols such as atomic force microscopy or shear rheology. Here we validate a direct method for acquiring elastic modulus of fibrin using a modified Hertz model for thin films. In this method,spherical indenters are positioned on top of the fibrin samples,generating an indentation depth that is then correlated with elastic modulus. Neurons function by transmitting electrical signals to one another and being able to assess the development of electrical signaling serves is an important verification step when engineering neural tissues. We then validated a protocol wherein the electrical activity of motor neural cultures is measured directly by a voltage sensitive dye and a microplate reader without causing damage to the cells. These protocols provide a non-destructive method for characterizing the mechanical and electrical properties of living spinal cord tissues using novel biosensing methods. View Publication

Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease,such as Parkinson's disease (PD),in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis,release,and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2,henceforth referred to as GIRK2),representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (textless10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites,and in the soma. Finally,neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models. View Publication

Although it is well established that women with germ-line mutations in the BRCA1 gene have a greatly increased lifetime incidence of breast and ovarian cancer,the molecular mechanisms responsible for this tissue-specific carcinogenesis remain undefined. The majority of these breast cancers are of the basal-like phenotype characterized by lack of expression of ER,PR,and ERBB2. Because this phenotype has been proposed to resemble that of normal breast stem cells,we examined the role of BRCA1 in human mammary stem cell fate. Using both in vitro systems and a humanized NOD/SCID mouse model,we demonstrate that BRCA1 expression is required for the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Knockdown of BRCA1 in primary breast epithelial cells leads to an increase in cells displaying the stem/progenitor cell marker ALDH1 and a decrease in cells expressing luminal epithelial markers and estrogen receptor. In breast tissues from women with germ-line BRCA1 mutations,but not normal controls,we detect entire lobules that,although histologically normal,are positive for ALDH1 expression but are negative for the expression of ER. Loss of heterozygosity for BRCA1 was documented in these ALDH1-positive lobules but not in adjacent ALDH1-negative lobules. Taken together,these studies demonstrate that BRCA1 plays a critical role in the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Because BRCA1 also plays a role in DNA repair,our work suggests that loss of BRCA1 may result in the accumulation of genetically unstable breast stem cells,providing prime targets for further carcinogenic events. View Publication

We observed that BMSCs (bone marrow stromal cells) from myeloma patients (myeloma BMSCs) were significantly stiffer than control BMSCs using a cytocompression device. The stiffness of myeloma BMSCs and control BMSCs was further increased upon priming by myeloma cells. Additionally,myeloma cells became stiffer when primed by myeloma BMSCs. The focal adhesion kinase activity of myeloma cells was increased when cells were on stiffer collagen gels and on myeloma BMSCs. This change in myeloma stiffness is associated with increased colony formation of myeloma cells and FAK activation when co-cultured with stiffer myeloma BMSCs or stiffer collagen. Additionally,stem cells of RPMI8226 cells became stiffer after priming by myeloma BMSCs,with concomitant increases of stem cell colony formation. These results suggest the presence of a mechanotransduction loop between myeloma cells and myeloma BMSCs to increase the stiffness of both types of cells via FAK activation. The increase of stiffness may in turn support the growth of myeloma cells and myeloma stem cells. View Publication

Human bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues like osteocytes,chondrocytes,and adipocytes in vivo and in vitro. The aim of this study was to investigate the in vitro differentiation of MSCs into cells of the endothelial lineage. MSCs were generated out of mononuclear bone marrow cells from healthy donors separated by density gradient centrifugation. Cells were characterized by flow cytometry using a panel of monoclonal antibodies and were tested for their potential to differentiate along different mesenchymal lineages. Isolated MSCs were positive for the markers CD105,CD73,CD166,CD90,and CD44 and negative for typical hematopoietic and endothelial markers. They were able to differentiate into adipocytes and osteocytes after cultivation in respective media. Differentiation into endothelial-like cells was induced by cultivation of confluent cells in the presence of 2% fetal calf serum and 50 ng/ml vascular endothelial growth factor. Laser scanning cytometry analysis of the confluent cells in situ showed a strong increase of expression of endothelial-specific markers like KDR and FLT-1,and immunofluorescence analysis showed typical expression of the von Willebrand factor. The functional behavior of the differentiated cells was tested with an in vitro angiogenesis test kit where cells formed characteristic capillary-like structures. We could show the differentiation of expanded adult human MSCs into cells with phenotypic and functional features of endothelial cells. These predifferentiated cells provide new options for engineering of artificial tissues based on autologous MSCs and vascularized engineered tissues. View Publication

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells,whereas mural cells are believed to derive from mesoderm,neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation,whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system. View Publication

The receptor for advanced glycation end products (RAGE) is produced either as a transmembrane or soluble form (sRAGE). Substantial evidence supports a role for RAGE and its ligands in disease. sRAGE is reported to be a competitive,negative regulator of membrane RAGE activation,inhibiting ligand binding. However,some reports indicate that sRAGE is associated with inflammatory disease. We sought to define the biological function of sRAGE on inflammatory cell recruitment,survival,and differentiation in vivo and in vitro. To test the in vivo impact of sRAGE,the recombinant protein was intratracheally administered to mice,which demonstrated monocyte- and neutrophil-mediated lung inflammation. We also observed that sRAGE induced human monocyte and neutrophil migration in vitro. Human monocytes treated with sRAGE produced proinflammatory cytokines and chemokines. Our data demonstrated that sRAGE directly bound human monocytes and monocyte-derived macrophages. Binding of sRAGE to monocytes promoted their survival and differentiation to macrophages. Furthermore,sRAGE binding to cells increased during maturation,which was similar in freshly isolated mouse monocytes compared with mature tissue macrophages. Because sRAGE activated cell survival and differentiation,we examined intracellular pathways that were activated by sRAGE. In primary human monocytes and macrophages,sRAGE treatment activated Akt,Erk,and NF-kappaB,and their activation appeared to be critical for cell survival and differentiation. Our data suggest a novel role for sRAGE in monocyte- and neutrophil-mediated inflammation and mononuclear phagocyte survival and differentiation. View Publication

USH2A mutations are the leading cause of autosomal recessive retinitis pigmentosa (RP),a progressive blinding disease marked by photoreceptor degeneration. Animal models fail to recapitulate the features of USH2A RP seen in humans,and its earliest pathogenic events remain unknown. Here,we established a human model of USH2A RP using retinal organoids derived from patient induced pluripotent stem cells and CRISPR-Cas9-engineered isogenic-USH2A−/− induced pluripotent stem cells. Methods: We assessed organoids for cellular,molecular,and morphological defects using serial live imaging and whole organoid and fixed section analyses. Results: Both patient-derived and isogenic-USH2A−/− organoids showed preferential rod photoreceptor loss followed by widespread degeneration,consistent with the clinical phenotype. Additionally,isogenic-USH2A−/− organoids showed early defects in proliferation and structure. Conclusions: Our findings suggest that molecular changes precede overt photoreceptor loss in USH2A RP,and pathogenesis may begin before clinical symptoms emerge. By defining early and late disease features,we provide new insight on the developmental origins of USH2A RP to guide therapeutic strategies. View Publication

Stem cell fate decisions,including proliferation,differentiation,morphological changes,and viability,are impacted by microenvironmental cues such as physical and biochemical signals. However,the specific impact of matrix elasticity on kidney cell development and function remains less understood due to the lack of models that can closely recapitulate human kidney biology. An established protocol to differentiate podocytes from human-induced pluripotent stem (iPS) cells provides a promising avenue to elucidate the role of matrix elasticity in kidney tissue development and lineage determination. In this study,we synthesized polyacrylamide hydrogels with different stiffnesses and investigated their ability to promote podocyte differentiation and biomolecular characteristics. We found that 3 kPa and 10 kPa hydrogels significantly support the adhesion,differentiation,and viability of podocytes. Differentiating podocytes on a more compliant (0.7 kPa) hydrogel resulted in significant cell loss and detachment. Further investigation of the mechanosensitive proteins yes-associated protein (YAP) and synaptopodin revealed nuanced molecular distinctions in cellular responses to matrix elasticity that may otherwise be overlooked if morphology and cell spreading alone were used as the primary metric for selecting matrices for podocyte differentiation. Specifically,hydrogels with kidney-like rigidities outperformed traditional tissue culture plates at modulating the molecular-level expression of active mechanosensitive proteins critical for podocyte health and function. These findings could guide the development of physiologically relevant platforms for kidney tissue engineering,disease modeling,and mechanistic studies of organ physiology and pathophysiology. Such advances are critical for realizing the full potential of in vitro platforms in accurately predicting human biological responses. View Publication

The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However,whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues,such as umbilical cord mesenchymal cells (UMCs),are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report,we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs),we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly,we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay,reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system,in T cell co-culture system as well. Furthermore,through whole genome expression microarray analysis,we showed that over 70 immune genes,including all members of HLA-I,were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation,thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine. View Publication

Human monoclonal antibodies against RSV have high potential for use as prophylaxis or therapeutic molecules,and they also can be used to define the structure of protective epitopes for rational vaccine design. In the past,however,isolation of human monoclonal antibodies was difficult and inefficient. Here,we describe contemporary methods for activation and proliferation of primary human memory B cells followed by cytofusion to non-secreting myeloma cells by dielectrophoresis to generate human hybridomas secreting RSV-specific monoclonal antibodies. We also provide experimental methods for screening human B cell lines to obtain RSV-specific lines,especially lines secreting neutralizing antibodies. View Publication

Dysregulated innate immune responses underlie multiple inflammatory diseases,but clinical translation of preclinical innate immunity research in mice is hampered by the difficulty of studying human inflammatory reactions in an in vivo context. We therefore sought to establish in vivo human inflammatory responses in NSG-QUAD mice that express four human myelopoiesis transgenes to improve engraftment of a human innate immune system. We reconstituted NSG-QUAD mice with human hematopoietic stem and progenitor cells (HSPCs),after which we evaluated human myeloid cell development and subsequent human responses to systemic and local lipopolysaccharide (LPS) challenges. NSG-QUAD mice already displayed engraftment of human monocytes,dendritic cells and granulocytes in peripheral blood,spleen and liver at 6 weeks after HSPC reconstitution,in which both classical,intermediate and non-classical monocytes were present. These huNSG-QUAD mice responded to intraperitoneal and intranasal LPS challenges with production of NF-κB-dependent human cytokines,a human type I interferon response,as well as inflammasome-mediated production of human IL-1β and IL-18. The latter were specifically abrogated by the NLRP3 inhibitor MCC950,while LPS-induced human monocyte death was not altered. Besides providing proof-of-principle for small molecule testing of human inflammatory reactions in huNSG-QUAD mice,this observation suggests that LPS-induced in vivo release of human NLRP3 inflammasome-generated cytokines occurs in a cell death-independent manner. HuNSG-QUAD mice are competent for the NF-κB,interferon and inflammasome effectors of human innate immunity,and can thus be utilized to investigate signaling mechanisms and pharmacological targeting of human inflammatory responses in an in vivo setting. View Publication