Adult Human Brain Neural Progenitor Cells (NPCs) and Fibroblast-Like Cells Have Similar Properties In Vitro but Only NPCs Differentiate into Neurons

The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC) culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP) of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia), and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs). These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5–6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments

Novel modular chimeric antigen receptor spacer for T cells derived from signal regulatory protein alpha Ig-like domains

Background: T cells equipped with chimeric antigen receptors (CAR) have shown remarkable efficacy in targeting B lineage malignancies. Improvement of the CAR structure is needed, however, with a view to developing flexibly modifiable spacers that are inert in interactions with unwanted cells. Specifically, binding to cells carrying receptors for IgG’s crystallizable fragment (FcR), that recognize IgG-derived domains in CARs is to be avoided.Methods: Two novel CARs targeting the CD19 antigen where the IgG1-CH2 and -CH3 domains were replaced with Ig-like domains from signal-regulatory protein α (SIRPα) were designed in silico. An IgG1-based CAR and a CAR lacking both SIRPα and IgG1 domains were used as comparators. The phenotype and memory phenotype of the expanded cells were analyzed by flow cytometry, and CAR T cell activation and cytotoxic efficacy were assessed in co-culture experiments in response to CD19+ target cells. Unwanted interactions with FcR-expressing myeloid cells were interrogated in co-culture assays with THP-1 monocytic cells.Results: T cells carrying the novel SIRPα-based CARs enacted potent in vitro cytotoxicity against CD19 positive B-lineage leukemia cells, comparable to traditional IgG1-based CAR T cells. Co-culture of IgG1-based CAR T cells with FcR-expressing THP-1 monocytic cells led to prominent cell surface expression of CD69 on T cells together with production of Interleukin (IL)-2 and Interferon-γ, and production of IL-1β, indicating activation of the T cells and monocytes, respectively. Longer co-culture led to killing of the monocytes. No signs of T cell nor monocyte activation were detected in co-cultures of SIRPα-based CAR T cells with THP-1 cells. Arming T cells with the SIRPα-based CARs favored differentiation towards CD4+ phenotype during expansion, while the effects on memory phenotype of the T cells were equivalent between the SIRPα- and IgG1-based CARs. In a pilot experiment, T cells modified with one of the SIRPα-based CARs showed dose dependent leukemia cell control.Conclusion: The novel SIRPα based spacers offer a suitable backbone for developing chimeric antigen receptors that evade the off-target binding to FcR while the cells retain a favorable memory phenotype and efficient cytotoxicity, establishing a promising candidate for future in vivo and clinical testing

Role of PrfA, internalins and ActA in host cell invasion by the bacterial pathogen Listeria monocytogenes

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

CAR T Cell Therapy: A Versatile Living Drug

After seeing a dramatic increase in the development and use of immunotherapy and precision medicine over the past few decades, oncological care now embraces the start of the adoptive cell therapy (ACT) era. This impulse towards a new treatment paradigm has been led by chimeric antigen receptor (CAR) T cells, the only type of ACT medicinal product to be commercialized so far. Brought about by an ever-growing understanding of cellular engineering, CAR T cells are T lymphocytes genetically modified with an appropriate DNA construct, which endows them with expression of a CAR, a fusion protein between a ligand-specific recognition domain, often an antibody-like structure, and the activating signaling domain of the T cell receptor. Through this genetic enhancement, CAR T cells are engineered from a cancer patient’s own lymphocytes to better target and kill their cancer cells, and the current amassed data on clinical outcomes point to a stream of bright developments in the near future. Herein, from concept design and present-day manufacturing techniques to pressing hurdles and bright discoveries around the corner, we review and thoroughly describe the state of the art in CAR T cell therapy

CAR T Cell Therapy: A Versatile Living Drug

After seeing a dramatic increase in the development and use of immunotherapy and precision medicine over the past few decades, oncological care now embraces the start of the adoptive cell therapy (ACT) era. This impulse towards a new treatment paradigm has been led by chimeric antigen receptor (CAR) T cells, the only type of ACT medicinal product to be commercialized so far. Brought about by an ever-growing understanding of cellular engineering, CAR T cells are T lymphocytes genetically modified with an appropriate DNA construct, which endows them with expression of a CAR, a fusion protein between a ligand-specific recognition domain, often an antibody-like structure, and the activating signaling domain of the T cell receptor. Through this genetic enhancement, CAR T cells are engineered from a cancer patient’s own lymphocytes to better target and kill their cancer cells, and the current amassed data on clinical outcomes point to a stream of bright developments in the near future. Herein, from concept design and present-day manufacturing techniques to pressing hurdles and bright discoveries around the corner, we review and thoroughly describe the state of the art in CAR T cell therapy

Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization

Abstract The immunoglobulin (Ig) superfamily members are involved in cell adhesion and migration, complex multistep processes that play critical roles in embryogenesis, wound healing, tissue formation, and many other processes, but their specific functions during embryonic development remain unclear. Here, we have studied the function of the immunoglobulin superfamily member 3 (IGSF3) by generating an Igsf3 knockout (KO) mouse model with CRISPR/Cas9-mediated genome engineering. By combining RNA and protein detection methodology, we show that during development, IGSF3 localizes to the neural crest and a subset of its derivatives, suggesting a role in normal embryonic and early postnatal development. Indeed, inactivation of Igsf3 impairs the ability of the vagal neural crest cells to migrate and normally innervate the intestine. The small intestine of Igsf3 KO mice shows reduced thickness of the muscularis externa and diminished number of enteric neurons. Also, misalignment of neurons and smooth muscle cells in the developing intestinal villi is detected. Taken together, our results suggest that IGSF3 functions contribute to the formation of the enteric nervous system. Given the essential role of the enteric nervous system in maintaining normal gastrointestinal function, our study adds to the pool of information required for further understanding the mechanisms of gut innervation and etiology behind bowel motility disorders

Efficacy and Safety of Glycosphingolipid SSEA-4 Targeting CAR-T Cells in an Ovarian Carcinoma Model

Chimeric antigen receptor (CAR) T-cell immunotherapies for solid tumors face critical challenges such as heterogeneous antigen expression. We characterized stage-specific embryonic antigen-4 (SSEA-4) cell-surface glycolipid as a target for CAR T-cell therapy. SSEA-4 is mainly expressed during embryogenesis but is also found in several cancer types making it an attractive tumor-associated antigen. Anti-SSEA-4 CAR-T cells were generated and assessed preclinically in vitro and in vivo for antitumor response and safety. SSEA-4 CAR-T cells effectively eliminated SSEA-4-positive cells in all the tested cancer cell lines, whereas SSEA-4-negative cells lines were not targeted. In vivo efficacy and safety studies using NSG mice and the high-grade serous ovarian cancer cell line OVCAR4 demonstrated a remarkable and specific antitumor response at all the CAR T-cell doses used. At high T-cell doses, CAR T cell-treated mice showed signs of health deterioration after a follow-up period. However, the severity of toxicity was reduced with a delayed onset when lower CAR T-cell doses were used. Our data demonstrate the efficacy of anti-SSEA-4 CAR T-cell therapy; however, safety strategies, such as dose-limiting and/or equipping CAR-T cells with combinatorial antigen recognition should be implemented for its potential clinical translation.Peer reviewe

A population of mitotically active cells from biopsy specimens show NPC-like characteristics.

<p>(<b>A</b>) Passage 3 of isolated cells grown as monolayer cultures in serum-free NPC proliferation media. The culture is composed of phase-bright dividing bipolar shaped cells and spontaneously forming and differentiating NSs. <b>(B)</b> Arrows indicate cells co-expressing Nestin (green) and GFAP (red). (<b>C-D</b>) Cells passaged and plated onto non-adhesive petri-dishes formed numerous NSs. (<b>E</b>) Represents a bright-field image of a differentiating NS and (<b>F</b>) shows these cells expressing βIII-tubulin (green). (<b>G-H</b>) βIII-tubulin and GFAP co-localized ‘asteron’ cells and (<b>I-J</b>) show single labelled neuronal MAP2 (arrow) and astroglial GFAP positive cells. (<b>K</b>) Pie graph showing the percentage of our human brain culture cases that generated AhNPCs (i.e. produced neurospheres, co-expressed Nestin and GFAP). The bar graph indicates the percentage of the AhNPC-positive cases that gave rise to βIII-tubulin, GFAP and MAP2 positive cells when differentiated. Scale: 100 µm.</p

Summary of the characteristics of brain-derived FbCs compared to the human lung-derived fibroblast cells.

<p>Arbitrary scale based on a combined observation of ICC, western blot and q-RT-PCR data.</p><p><b>−</b>  =  not detected, <b>+</b>  =  small, <b>++</b>  =  medium, <b>+++</b>  =  high expression.</p

The FbCs isolated from biopsy specimens show NPC-like properties under NPC culture conditions but failed to differentiate into functional neurons.

<p>(<b>A</b>) Bright-field (BF) image showing the distinct morphology of the FbCs. They exhibited a flat and diffuse cytoplasm and were generally phase-dark. These cells showed characteristics of fibroblasts, as they lack the expression of neuronal βIII-tubulin (<b>B</b>), but had high levels of collagen synthesizing enzyme prolyl-4-hydroxylase (P4H; <b>D</b>) and Vimentin (<b>F</b>). However, they also expressed stem cell-like markers such as Nestin (<b>C</b>) and Sox-2 (<b>E</b>). Furthermore, when cultured under neurosphere-forming conditions, FbCs also formed spheres (<b>G</b>) that could be passaged for at least 2 – 3 passages. When FbCs were cultured in NPC culture conditions, their morphology changed to phase-bright polar shaped cells (<b>H</b>) and started expressing high levels of βIII-tubulin (<b>I</b>) and up-regulated their expression of Nestin (<b>J</b>). In agreement with immunostaining results, quantitative image analysis revealed NPC conditions gradually increased the percentage of FbCs expressing Nestin and βIII-tubulin at a detectable level (<b>K</b>). When compared to the control astrocytic culture condition, these differences were significant (P&lt;0.01). Q-RT-PCR data also showed βIII-tubulin mRNA levels increased by over 10 fold by 14 days of exposure to NPC conditions (<b>L</b>; P&lt;0.01). Due to the relatively high basal levels of Nestin in the control cells, the relative increase in Nestin was only 1.5 fold, but this still reached significance (<b>L</b>; P&lt;0.01). Finally, western blots validated many of the above observations, as in control conditions, they showed βIII-tubulin at near un-detectable levels, but increased significantly during the first 3 days in NPC media and continued to increase till day 7 (<b>M</b>). Sox-2 and Vimentin were highly expressed in control and NPC media-induced FbCs (<b>M</b>). These results were consistently observed in all the cases tested (n = 5). Scale: 100 µm.</p