Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (<P10) of the cell lines revealing there were no genetic drift over the passages. Karyotyping also revealed no chromosomal anomalies. Creation of these cell lines can standardize experimental approaches, eliminate subject to subject variabilities, and benefit the reproductive biological studies on pregnancies by using these cells.

Molecular Mechanisms of Anticancer Activity of N-Glycosides of Indolocarbazoles LCS-1208 and LCS-1269

Novel indolocarbazole derivatives named LCS were synthesized by our research group. Two of them were selected as the most active anticancer agents in vivo. We studied the mechanisms of anticancer activity in accordance with the previously described effects of indolocarbazoles. Cytotoxicity was estimated by MTT assay. We analyzed LCS-DNA interactions by circular dichroism in cholesteric liquid crystals and fluorescent indicator displacement assay. The effect on the activity of topoisomerases I and II was studied by DNA relaxation assay. Expression of interferon signaling target genes was estimated by RT-PCR. Chromatin remodeling was analyzed–the effect on histone H1 localization and reactivation of epigenetically silenced genes. LCS-induced change in the expression of a wide gene set was counted by means of PCR array. Our study revealed the cytotoxic activity of the compounds against 11 cancer cell lines and it was higher than in immortalized cells. Both compounds bind DNA; binding constants were estimated—LCS-1208 demonstrated higher affinity than LCS-1269; it was shown that LCS-1208 intercalates into DNA that is typical for rebeccamycin derivatives. LCS-1208 also inhibits topoisomerases I and IIα. Being a strong intercalator and topoisomerase inhibitor, LCS-1208 upregulates the expression of interferon-induced genes. In view of LCSs binding to DNA we analyzed their influence on chromatin stability and revealed that LCS-1269 displaces histone H1. Our analysis of chromatin remodeling also included a wide set of epigenetic experiments in which LCS-1269 demonstrated complex epigenetic activity. Finally, we revealed that the antitumor effect of the compounds is based not only on binding to DNA and chromatin remodeling but also on alternative mechanisms. Both compounds induce expression changes in genes involved in neoplastic transformation and target genes of the signaling pathways in cancer cells. Despite of being structurally similar, each compound has unique biological activities. The effects of LCS-1208 are associated with intercalation. The mechanisms of LCS-1269 include influence on higher levels such as chromatin remodeling and epigenetic effects.

FAM83A is a Potential Biomarker for Breast Cancer Initiation

Background: Family with sequence similarity 83 member A (FAM83A) presents oncogenic properties in several cancers including breast cancer (BC). Recently, we reported FAM83A overexpression in normal breast tissues from women at high risk of breast cancer. We now hypothesize that FAM83A is a key factor in BC initiation. Methods: Immunohistochemical staining was used to evaluate FAM83A protein levels in both a normal breast tissue microarray (TMA, N=411) and a breast tumor TMA (N=349). EGFR staining and its correlation with FAM83A expression were also assessed. Lentivirus-mediated manipulation of FAM83A expression in primary and hTERT-immortalized breast epithelial cells was employed. Biological and molecular alterations upon FAM83A overexpression/downregulation and FAM83A’s interaction partners were investigated.Results: TMA analysis revealed a 1.5-fold increase in FAM83A expression level in BC cases as compared with normal breast tissues (p<0.0001). FAM83A protein expression was directly correlated with EGFR level in both normal and BC tissues. In in vitro assays, exogenous expression of FAM83A in either primary or immortalized breast epithelial cells promoted cell viability and proliferation. Additionally, Ingenuity Pathway Analysis (IPA) revealed that in normal cells FAM83A is involved in cellular morphology and metabolism. Mass spectrometry analysis identified DDX3X and LAMB3 as potential FAM83A interaction partners in primary cells, while we detected FAM83A interaction with cytoskeleton reorganization factors, including LIMA1, MYH10, PLEC, MYL6 in the immortalized cells.Conclusions: This study shows that FAM83A promotes metabolic activation in primary epithelial cells and survival in immortalized cells. These findings support its role in early breast oncogenesis.

Establishment and genetic characterization of cell lines derived from proliferating nasal polyps and sinonasal inverted papillomas

To better understand the pathogenesis of nasal polyps (NPs) and sinonasal inverted papillomas (SIPs), we aimed to establish cell lines from fresh tissues of NPs and SIPs and characterize them. Primary cell cultures were obtained from two NP tissues (NP2 and NP3) and one SIP tissue (IP4). All the cells were polygonal in shape, expressed cytokeratin 14, and had normal diploid chromosome status. HPV58 DNA was detected in NP3. To obtain immortal primary cells, NP2 and IP4 cells were transduced with a combination of mutant CDK4, cyclinD1 and TERT. These cells were thereafter named NP2/K4DT and IP4/K4DT, respectively. HPV58-positive NP3 cells were transduced with TERT alone, the resulting cells named NP3/T. Phenotypic and genotypic identity of original tissues and derived cells was investigated. All the cell cultures with transgenes were confirmed to be derived from their parental cells and primary tumor tissues by analysis of short tandem repeats (STR) and maintained in vitro growth, genetic profiles and gene expression characteristics of the primary cells. These virtually immortalized cells, as well as the primary cells, have potential as in vitro models for studying the pathogenesis of NPs and SIPs and for preclinical study to develop new therapeutic agents.

Oleogel Formulations for the Topical Delivery of Betulin and Lupeol in Skin Injuries—Preparation, Physicochemical Characterization, and Pharmaco-Toxicological Evaluation

The skin integrity is essential due to its pivotal role as a biological barrier against external noxious factors. Pentacyclic triterpenes stand as valuable plant-derived natural compounds in the treatment of skin injuries due to their anti-inflammatory, antioxidant, antimicrobial, and healing properties. Consequently, the primary aim of the current investigation was the development as well as the physicochemical and pharmaco-toxicological characterization of betulin- and lupeol-based oleogels (Bet OG and Lup OG) for topical application in skin injuries. The results revealed suitable pH as well as organoleptic, rheological, and textural properties. The penetration and permeation of Bet and Lup oleogels through porcine ear skin as well as the retention of both oleogels in the skin were demonstrated through ex vivo studies. In vitro, Bet OG and Lup OG showed good biocompatibility on HaCaT human immortalized cells. Moreover, Bet OG exerted a potent wound-healing property by stimulating the migration of the HaCaT cells. The in ovo results demonstrated the non-irritative potential of the developed formulations. Additionally, the undertaken in vivo investigation indicated a positive effect of oleogels treatment on skin parameters by increasing skin hydration and decreasing erythema. In conclusion, oleogel formulations are ideal for the local delivery of betulin and lupeol in skin disorders.

Neuroprotective Potential of a Small Molecule RET Agonist in Cultured Dopamine Neurons and Hemiparkinsonian Rats

Background: Parkinson’s disease (PD) is a progressive neurological disorder where loss of dopamine neurons in the substantia nigra and dopamine depletion in the striatum cause characteristic motor symptoms. Currently, no treatment is able to halt the progression of PD. Glial cell line-derived neurotrophic factor (GDNF) rescues degenerating dopamine neurons both in vitro and in animal models of PD. When tested in PD patients, however, the outcomes from intracranial GDNF infusion paradigms have been inconclusive, mainly due to poor pharmacokinetic properties. Objective: We have developed drug-like small molecules, named BT compounds that activate signaling through GDNF’s receptor, the transmembrane receptor tyrosine kinase RET, both in vitro and in vivo and are able to penetrate through the blood-brain barrier. Here we evaluated the properties of BT44, a second generation RET agonist, in immortalized cells, dopamine neurons and rat 6-hydroxydopamine model of PD. Methods: We used biochemical, immunohistochemical and behavioral methods to evaluate the effects of BT44 on dopamine system in vitro and in vivo. Results: BT44 selectively activated RET and intracellular pro-survival AKT and MAPK signaling pathways in immortalized cells. In primary midbrain dopamine neurons cultured in serum-deprived conditions, BT44 promoted the survival of the neurons derived from wild-type, but not from RET knockout mice. BT44 also protected cultured wild-type dopamine neurons from MPP +-induced toxicity. In a rat 6-hydroxydopamine model of PD, BT44 reduced motor imbalance and could have protected dopaminergic fibers in the striatum. Conclusion: BT44 holds potential for further development into a novel, possibly disease-modifying therapy for PD.

CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells

CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR (http://z.umn.edu/spliceR) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.

Investigating the effects of IDO1, PTGS2, and TGF-β1 overexpression on immunomodulatory properties of hTERT-MSCs and their extracellular vesicles

The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawbacks have resulted in a strategic shift towards the application of MSC-derived cell-free products such as extracellular vesicles (EVs). Recent reports revealed that functional properties of MSCs, including EV secretion patterns, correlate with microenvironmental cues. These findings highlight the urgent need for defining the optimal circumstances for EV preparation. Considering the limitations of primary cells, we employed immortalized cells as an alternative source to prepare therapeutically sufficient EV numbers. Herein, the effects of different conditional environments are explored on human TERT-immortalized MSCs (hTERT-MSCs). The latter were transduced to overexpress IDO1, PTGS2, and TGF-β1 transgenes either alone or in combination, and their immunomodulatory properties were analyzed thereafter. Likewise, EVs derived from these various MSCs were extensively characterized. hTERT-MSCs-IDO1 exerted superior inhibitory effects on lymphocytes, significantly more than hTERT-MSCs-IFN-γ. As such, IDO1 overexpression promoted the immunomodulatory properties of such enriched EVs. Considering the limitations of cell therapy like tumor formation and possible immune responses in the host, the results presented herein might be considered as a feasible model for the induction of immunomodulation in off-the-shelf and cell-free therapeutics, especially for autoimmune diseases.

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased β-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.