Establishment of a human induced pluripotent stem cell neuronal model for identification of modulators of A53T α-synuclein levels and aggregation

Inhibiting formation or promoting degradation of α-synuclein aggregates are among the therapeutical approaches under investigation as disease-modifying treatment strategies for Parkinson’s disease. To support these developments, several in vitro models based on seeded α-synuclein aggregation have been established in immortalized cell lines and murine primary neurons. Here, we report on a humanized model with a reproducibility and throughput that enables its use in supporting target identification and validation in pharmacological research. A human induced pluripotent stem cell (iPSC) line was genetically modified to express HA-tagged α-synuclein with the point mutation in position 53 from Alanine to Threonine (A53T) under an inducible system and differentiated into cortical neurons expressing neuronal markers and exhibiting spontaneous activity. Intracellular α-synuclein aggregation was triggered by exposure to exogenous added fibrillated recombinant wild-type human α-synuclein fibrils91 and demonstrated by several endpoints; the formation of Triton-insoluble SDS-soluble α-synuclein, biochemically in a fluorescence resonance energy transfer based aggregation assay and by immunocytochemistry of phosphorylated α-synuclein positive puncta. We demonstrate the feasibility of upscaling the iPSC neuron production for drug discovery and that the model has a suitable dynamic range allowing for both detection of increased and decreased α-synuclein aggregation. Moreover, gene modulation is feasible using siRNAs, making the model suitable for genetic screening for modulators of α-synuclein aggregation. Data on effects of USP8, USP13 and USP9X knockdown on α-synuclein expression and aggregation contradicts published data from immortalized cell lines and murine systems. This highlight the importance of including humanized neuronal models in the confirmation of biological mechanisms in specific variations of Parkinson’s disease.

Isolation of Primary Mouse Pulmonary Microvascular Endothelial Cells and Generation of an Immortalized Cell Line to Obtain Sufficient Extracellular Vesicles

Pulmonary microvascular endothelial cells (PMECs) and the extracellular vesicles (EVs) derived from PMECs participate in maintaining pulmonary homeostasis and mediating the inflammatory response. However, obtaining a high-purity population of PMECs and their EVs from mouse is still notoriously difficult. Herein we provide a method to isolate primary mouse PMECs (pMPMECs) and to transduce SV40 lentivirus into pMPMECs to establish an immortalized cell line (iMPMECs), which provides sufficient quantities of EVs for further studies. pMPMECs and iMPMECs can be identified using morphologic criteria, a phenotypic expression profile (e.g., CD31, CD144, G. simplicifolia lectin binding), and functional properties (e.g., Dil-acetylated low-density protein uptake, Matrigel angiogenesis). Furthermore, pMPMEC–EVs and iMPMEC–EVs can be identified and compared. The characteristics of pMPMEC–EVs and iMPMEC–EVs are ascertained by transmission electron microscopy, nanoparticle tracking analysis, and specific protein markers. iMPMECs produce far more EVs than pMPMECs, while their particle size distribution is similar. Our detailed protocol to isolate and immortalize MPMECs will provide researchers with an in vitro model to investigate the specific roles of EVs in pulmonary physiology and diseases.

Establishment and Functional Characterization of Immortalized Rabbit Dermal Papilla Cell Lines

Background Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in the HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. Method In this study, we introduced SV40LT into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DP cell lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). Result These cell lines displayed early passage morphology and displayed high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA ,IGF1, ALPL, FGF2, BMP2 and TGFβ2; α-SMA and VIM protein), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. Conclusion The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

Factor quinolinone inhibitors alter cell morphology and motility by destabilizing interphase microtubules

Factor quinolinone inhibitors are promising anti-cancer compounds, initially characterized as specific inhibitors of the oncogenic transcription factor LSF (TFCP2). These compounds exert anti-proliferative activity at least in part by disrupting mitotic spindles. Herein, we report additional interphase consequences of the initial lead compound, FQI1, in two telomerase immortalized cell lines. Within minutes of FQI1 addition, the microtubule network is disrupted, resulting in a substantial, although not complete, depletion of microtubules as evidenced both by microtubule sedimentation assays and microscopy. Surprisingly, this microtubule breakdown is quickly followed by an increase in tubulin acetylation in the remaining microtubules. The sudden breakdown and partial depolymerization of the microtubule network precedes FQI1-induced morphological changes. These involve rapid reduction of cell spreading of interphase fetal hepatocytes and increase in circularity of retinal pigment epithelial cells. Microtubule depolymerization gives rise to FH-B cell compaction, as pretreatment with taxol prevents this morphological change. Finally, FQI1 decreases the rate and range of locomotion of interphase cells, supporting an impact of FQI1-induced microtubule breakdown on cell motility. Taken together, our results show that FQI1 interferes with microtubule-associated functions in interphase, specifically cell morphology and motility.

Effect of Cell Cycle on Cell Surface Expression of Voltage-Gated Sodium Channels and Na+,K+-ATPase

Voltage-gated sodium channels (VGSCs) are the target for many therapies. Variation in membrane potential occurs throughout the cell cycle, yet little attention has been devoted to VGSCs and Na+,K+-ATPase in the cell cycle. We hypothesized that in addition to doubling DNA and cell membrane in anticipation of cell division, there should be a doubling of VGSCs and Na+,K+-ATPase compared to non-dividing cells. We tested this hypothesis in eight immortalized cell lines by correlating immunocytofluorescent labeling of VGSCs or Na+,K+-ATPase, with propidium iodide or DAPI fluorescence using flow cytometry. Cell surface expression of VGSCs during phases S through M was double that seen during phases G0 - G1. By contrast, Na+,K+-ATPase expression increased only 1.5-fold. The increases were independent of baseline expression of channels or pumps. The variation in VGSC and Na+,K+-ATPase expression has implications for both our understanding of sodium's role in controlling the cell cycle and variability of treatments targeted at these components of the Na+ handling system.

Human iPSC-Derived Neurons as A Platform for Deciphering the Mechanisms behind Brain Aging

With an increased life expectancy among humans, aging has recently emerged as a major focus in biomedical research. The lack of in vitro aging models—especially for neurological disorders, where access to human brain tissues is limited—has hampered the progress in studies on human brain aging and various age-associated neurodegenerative diseases at the cellular and molecular level. In this review, we provide an overview of age-related changes in the transcriptome, in signaling pathways, and in relation to epigenetic factors that occur in senescent neurons. Moreover, we explore the current cell models used to study neuronal aging in vitro, including immortalized cell lines, primary neuronal culture, neurons directly converted from fibroblasts (Fib-iNs), and iPSC-derived neurons (iPSC-iNs); we also discuss the advantages and limitations of these models. In addition, the key phenotypes associated with cellular senescence that have been observed by these models are compared. Finally, we focus on the potential of combining human iPSC-iNs with genome editing technology in order to further our understanding of brain aging and neurodegenerative diseases, and discuss the future directions and challenges in the field.

179 CD8+CD69+ Expanded Tumor Infiltrating Lymphocytes from Soft Tissue Sarcoma Have Increased Tumor-Specific Functional Capacity

BackgroundAdoptive cell therapy (ACT) utilizing tumor infiltrating lymphocytes (TIL) has demonstrated durable responses in patients with metastatic melanoma and offers potential for other solid tumors. Preclinical experience with expanded TIL from soft tissue sarcoma (STS) demonstrates less frequent tumor-specific reactivity compared to melanoma samples, limiting the potential for efficacy.1 We hypothesized that CD69+ TIL have increased tumor-specific reactivity, which can be manipulated in culture, thereby offering an opportunity to enhance the antitumor effect of this cellular immunotherapy product.MethodsPatients were enrolled on an IRB-approved protocol and TIL were expanded from fresh surgical specimens. After enzymatic digestion, tumor single cell suspensions were cultured in media containing 10% human serum and IL-2 (6000IU/mL). Expanded TIL were then enriched for CD8+ using magnetic bead isolation and CD69+ by flow cytometry cell sorting (FACS). After co-culture with autologous tumor digest, functional capacity was compared between bulk TIL and enriched TIL by evaluation of IFN-gamma (IFNg) and Granzyme B (GzB) secretion. Capacity for direct tumor cytotoxicity was assessed by Cr51 assay after co-culture of autologous immortalized cell lines with expanded TIL subpopulations after enrichment.ResultsFollowing co-culture with autologous tumor digest, CD69+ TIL demonstrated increased IFNg secretion compared to CD69- TIL in 6 samples (1.4–4.2x, p<0.05). CD8+ enriched TIL (75% compared to bulk) had higher relative IFNg secretion in both CD69+ and CD69- subsets (4.2 and 5.8x, respectively, p<0.001). Maximal IFNg secretion was seen from TIL that were both CD69+ sorted and CD8+ enriched, demonstrating an synergistic effect (16.3x vs Bulk CD69-, 4.2x vs Bulk CD69+, 2.8x vs CD8 enriched CD69- ; p<0.001). Functional capacity was also assessed by GzB secretion with similar results. CD69+ TIL had increased relative secretion (1.8–2.2x) compared to CD69- TIL (p< 0.01). CD8+ enriched TIL had increased relative GzB secretion in both CD69- and CD69+ sorted fractions (1.4x, 1.2x, respectively, p<.05). CD69+ sorted and CD8+ enriched TIL demonstrated an additive effect (2.6x vs Bulk CD69-, p<0.01; 1.2x vs Bulk CD69+, p<0.05; 1.8x vs CD8 enriched CD69-, p<0.01). CD8+ enriched CD69+ sorted TIL had greater relative cytotoxicity (3x, p<0.05) at 40:1 E:T ratio against autologous tumor cell lines compared to bulk expanded TIL (figure 1).Abstract 179 Figure 1Functional capacity of CD69+ TIL is demonstrated by increased secretion of GzB (A) and IFNg (B) after co-culture with autologous tumor digest. CD69+ TIL have greater cytotoxicity against autologous immortalized cell lines compared to bulk TIL at 40:1 E:T ratio (C).ConclusionsTIL expanded from STS demonstrate greater tumor-specific functional capacity and cytotoxicity after CD8 enrichment and CD69+ FACS compared to bulk expanded TIL. These data validate the strategy to enhance CD8+CD69+ TIL during culture to yield a more efficacious cellular immunotherapy product.AcknowledgementsThis work was funded by NIH K08CA252642Trial Registration n/aReference1. Mullinax JE, Hall M, Beatty M, Weber AM, Sannasardo Z, Svrdlin T, Hensel J, Bui M, Richards A, Gonzalez RJ, Cox CA, Kelley L, Mulé JJ, Sarnaik AA, Pilon-Thomas S. Expanded Tumor-infiltrating Lymphocytes From Soft Tissue Sarcoma Have Tumor-specific Function. J Immunother 2021 Feb-Mar 01;44(2):63–70.Ethics ApprovalAbstract cites IRB-approved protocol in methods section.Consent n/a

An Economic Approach to Energy Budgets: How Many Resources Should Living Organisms Spare?

Ramsey&rsquo;s economic theory of saving (RTS) estimates how much of its commodities a nation should save to safeguard the well-being of future generations. Since RTS retains many attractive qualities such as simplicity, strength, breadth and generality, here we ask if it would be useful to investigate biophysical issues. Specifically, we focus on a biological topic that lends itself as a backdrop for the study of the imbalance between intake and expenditure, i.e., the evaluation of the multicellular living organisms&rsquo; energetic requirements and constraints. Our problem is to find at each time the optimum distribution and the right balance of the cellular energy budget between consumption and storage: how much must a living organism spare to increase its chances of survival over long periods? Suggesting how to find the optimum allocation of the available energy between expenditure and saving at each time, RTS approaches to biological energy budgets may have a wide range of experimental applications, such as: a) optimization of the long-term survival chances of either immortalized cell cultures, or beneficial bacterial colonies and exogenous probiotic mixtures; b) eradication of detrimental biofilms, such as, e.g., heart valves&rsquo; Streptococcus colonies; c) novel anti-stress and anti-ageing strategies.

Functional analysis of a common BAG3 allele associated with protection from heart failure

Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism for such protection remains elusive. One of the variants in this allele block is coding, changing cysteine to arginine at position 151 of BAG3 (rs2234962-BAG3C151R). Here, we use induced pluripotent stem cells (iPSC) to test if the BAG3C151R variant alters protein and cellular function in human cardiac myocytes. Quantitative protein interaction network analysis identified specific changes in BAG3C151R protein interaction partners in cardiomyocytes but not in iPSCs or an immortalized cell line. Knockdown of BAG3 interacting factors in cardiomyocytes followed by myofibrillar analysis revealed that BAG3C151R associates more strongly with proteins involved in the maintenance of myofibrillar integrity. Finally, we demonstrate that cardiomyocytes expressing the BAG3C151R variant have improved response to proteotoxic stress in an allele dose-dependent manner. This study suggests that the BAG3C151R variant increases cardiomyocyte protection from stress by enhancing the recruitment of factors critical to the maintenance of myofibril integrity, hinting that this variant could be responsible for the cardioprotective effect of the haplotype block. By revealing specific changes in preferential binding partners of the BAG3C151R protein variant, we also identify potential targets for the development of novel cardioprotective therapies.