Three-Dimensional Biohybrid StarPEG-Heparin Hydrogel Cultures for Modeling Human Neuronal Development and Alzheimer's Disease Pathology.

In this chapter, we present the methodology currently used in our laboratory to generate a starPEG-MMP (starPEG)- and heparin maleimide HM06 (heparin)-based 3D cell culture system, in a hydrogel, that can be used to study human neuronal development and Alzheimer's disease (AD) pathology. A 3D cell culture system can mimic the in vivo cellular environment better than a 2D format, in which these cells exhibit neural network formation, electrophysiological activity, tissue-specific extracellular matrix (ECM) deposition, and neurotransmitter responsiveness. When treated with amyloid beta-42 (Aß42) peptides, this system recapitulates many of the pathological effects of AD, including reduced neural stem cell proliferation, impaired neuronal network formation, dystrophic axonal ends, synaptic loss, failure to deposit ECM, elevated tau hyperphosphorylation, and formation of neurofibrillary tangles. Culturing human primary cortical astrocyte (pHA)- or induced pluripotent stem cell (iPSC)-derived human neural stem cells in this biohybrid hydrogel system has led to the discovery of novel regulatory pathways underlying neurodegenerative pathology in different phases of AD.

GATA3 Promotes the Neural Progenitor State but Not Neurogenesis in 3D Traumatic Injury Model of Primary Human Cortical Astrocytes.

Astrocytes are abundant cell types in the vertebrate central nervous system and can act as neural stem cells in specialized niches where they constitutively generate new neurons. Outside the stem cell niches, however, these glial cells are not neurogenic. Although injuries in the mammalian central nervous system lead to profound proliferation of astrocytes, which cluster at the lesion site to form a gliotic scar, neurogenesis does not take place. Therefore, a plausible regenerative therapeutic option is to coax the endogenous reactive astrocytes to a pre-neurogenic progenitor state and use them as an endogenous reservoir for repair. However, little is known on the mechanisms that promote the neural progenitor state after injuries in humans. Gata3 was previously found to be a mechanism that zebrafish brain uses to injury-dependent induction of neural progenitors. However, the effects of GATA3 in human astrocytes after injury are not known. Therefore, in this report, we investigated how overexpression of GATA3 in primary human astrocytes would affect the neurogenic potential before and after injury in 2D and 3D cultures. We found that primary human astrocytes are unable to induce GATA3 after injury. Lentivirus-mediated overexpression of GATA3 significantly increased the number of GFAP/SOX2 double positive astrocytes and expression of pro-neural factor ASCL1, but failed to induce neurogenesis, suggesting that GATA3 is required for enhancing the neurogenic potential of primary human astrocytes and is not sufficient to induce neurogenesis alone.

3D Culture Method for Alzheimer's Disease Modeling Reveals Interleukin-4 Rescues Aß42-Induced Loss of Human Neural Stem Cell Plasticity.

Neural stem cells (NSCs) constitute an endogenous reservoir for neurons that could potentially be harnessed for regenerative therapies in disease contexts such as neurodegeneration. However, in Alzheimer's disease (AD), NSCs lose plasticity and thus possible regenerative capacity. We investigate how NSCs lose their plasticity in AD by using starPEG-heparin-based hydrogels to establish a reductionist 3D cell-instructive neuro-microenvironment that promotes the proliferative and neurogenic ability of primary and induced human NSCs. We find that administration of AD-associated Amyloid-ß42 causes classical neuropathology and hampers NSC plasticity by inducing kynurenic acid (KYNA) production. Interleukin-4 restores NSC proliferative and neurogenic ability by suppressing the KYNA-producing enzyme Kynurenine aminotransferase (KAT2), which is upregulated in APP/PS1dE9 mouse model of AD and in postmortem human AD brains. Thus, our culture system enables a reductionist investigation of regulation of human NSC plasticity for the identification of potential therapeutic targets for intervention in AD.

Stromal-Based Signatures for the Classification of Gastric Cancer.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

Regulation of human dihydrofolate reductase activity and expression.

Dihydrofolate reductase (DHFR) enzyme catalyzes tetrahydrofolate regeneration by reduction of dihydrofolate using NADPH as a cofactor. Tetrahydrofolate and its one carbon adducts are required for de novo synthesis of purines and thymidylate, as well as glycine, methionine and serine. DHFR inhibition causes disruption of purine and thymidylate biosynthesis and DNA replication, leading to cell death. Therefore, DHFR has been an attractive target for chemotherapy of many diseases including cancer. Over the following years, in order to develop better antifolates, a detailed understanding of DHFR at every level has been undertaken such as structure-functional analysis, mechanisms of action, transcriptional and translation regulation of DHFR using a wide range of technologies. Because of this wealth of information created, DHFR has been used extensively as a model system for enzyme catalysis, investigating the relations between structure in-silico structure-based drug design, transcription from TATA-less promoters, regulation of transcription through the cell cycle, and translational autoregulation. In this review, the current understanding of human DHFR in terms of structure, function and regulation is summarized.

Immunization with UV-induced apoptotic cells generates monoclonal antibodies against proteins differentially expressed in hepatocellular carcinoma cell lines.

Early and differential diagnosis of hepatocellular carcinoma (HCC) requires sensitive and specific tissue and serum markers. On the other hand, proteins involved in tumorigenesis are extensively modulated on exposure to apoptotic stimuli, including ultraviolet (UVC) irradiation. Hence, we generated monoclonal antibodies by using UVC-irradiated apoptotic cells of an HCC cell line, HUH7, aiming to explore proteins differentially expressed in tumors and apoptosis. We obtained 18 hybridoma clones recognizing protein targets in apoptotic HUH7 cells, and clone 6D5 was chosen for characterization studies because of its strong reactivity in cell-ELISA assay. Subtype of the antibody was IgG3 (kappa). Targets of 6D5 antibody were found to be abundantly expressed in all HCC cell lines except FLC4, which resembles normal hepatocytes. We also observed the secretion of 6D5 ligands by some of the HCC cell lines. Moreover, cellular proteins recognized by the antibody displayed a late upregulation in UVC-induced apoptotic cells. We concluded that 6D5 target proteins are modulated in liver tumorigenesis and apoptotic processes. We therefore propose the validation of our antibody in tissue and serum samples of HCC patients to assess its potential use for the early diagnosis of HCC and to understand the role of 6D5 ligands in liver carcinogenesis.