Pure Populations Research Articles

Nestin gene expression in stromal precursor cells from the human bone marrow

INTRODUCTION: The hierarchy of stromal progenitors from the bone marrow is poorly characterized; multipotent mesenchymal stromal cells and colony-forming units of fibroblasts are isolated in culture. Mesenchymal stem cells do not have a unique combination of surface antigens, making it difficult to obtain the pure population. The expression of the nestin gene is often used as a marker of these cells.
 AIM: To evaluate the level of expression of the nestin gene in multipotent mesenchymal stromal cells and in colony-forming units of fibroblasts and to characterize the change in its expression during the transition from oligopotent progenitor cells to monopotent ones.
 MATERIALS AND METHODS: Stromal progenitors were analyzed in bone marrow samples from 19 donors by standard methods. A total of 296 individual clones of fibroblast colony-forming units were obtained from the same bone marrow samples. The cells were analyzed for the ability to differentiate toward the adipogenic and osteogenic lineages. Relative expression level of nestin gene was analyzed in all cells.
 RESULTS: Mean relative expression level of nestin did not differ significantly in multipotent mesenchymal stromal cells (0.410.13) and in the total population of colony-forming units of fibroblasts (0.240.05). In individual clones of colony-forming units of fibroblasts, nestin expression was not significantly higher than in the total population (0.310.04). When analyzing colony-forming units of fibroblasts differing in their differentiation potential, the highest expression of nestin was found in the group of monopotent osteogenic progenitors, while its expression was significantly lower in oligopotent progenitors.
 CONCLUSION: Nestin gene expression in mesenchymal stromal progenitors from the bone marrow is not specific for mesenchymal stem cells and cannot be used as a unique marker of this cell type. According to our data, a high level of nestin expression rather identifies monopotent osteogenic progenitors.

Laser Capture Microdissection - A Breakthrough Technology in Head and Neck Diagnostics

Laser capture microdissection (LCM) is a technique in which, from a heterogenous tissue specimen some pure cell populations are isolated. This procedure can capture and target the desired number of cells that are required. A low energy laser pulse is used in this technique. Apart from systemic applications LCM can also be applied in the field of dentistry and pathology. Bangladesh Journal of Medical Science Vol.22 (Special Issue) 2023 p.58-62

Immunomodulatory effects of β-defensin 2 on tumor associated macrophages induced antitumor function in breast cancer

BackgroundTumor-associated macrophages (TAMs) express cytokines and chemokines that can suppress antitumor immunity and promote tumor progression. The immunomodulatory and antitumor function of β-defensin 2 is still unclear, despite the evidence of infection response. We previously reported that β-defensin 2 modulates immunomodulatory and their antitumor function of macrophages in breast cancer. We investigate the association between β-defensin 2 and TAMs and determined the role in tumor-promoting attributes of TAMs reversal of phenotype in tumor regression. MethodsSwiss albino mice and C127i breast cancer cell line was used in this study. C127i conditioned media was prepared and generated macrophage-derived TAMs to study antitumor function. Flow cytometry was performed for phenotype identification of macrophages and TAMs. MTT assay was performed to estimate cytotoxicity and dose optimization of β-defensin 2. Oxidative stress was analyzed by H2O2 and NO estimation, and qPCR was performed for iNOS, cytokines and chemokines expression. ResultsPEC harvested macrophages were characterized by flow-cytometry using F4/80, CD11c antibodies with 98% pure population of macrophages and cultured in C127i conditioned media for 7 days. TAMs markers were estimated, and it was found that 98% expression of F4/80, CD-206, and CD-115 expression compared to macrophages. Purified 100 ng/ml of β-defensin 2 was used to stimulate the TAMs population was viable, which was confirmed by cell viability assay. ROS levels decreased in TAMs treated with β-defensin 2 compared to control group. Interleukins (ILs)-6, 10, and 3, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β and chemokine ligand (CXCL)-1, 5 and 15, chemokine ligand (CCL)-24 and 5 decreased drastically compared to control. ConclusionThis is the first report of β-defensin 2 on TAMs to elucidate the immunomodulatory and anti-tumor function. It was found that the cytokines, chemokines, and reactive oxygen species (ROS) expression pliably changed which facilitates tumor regression. β-defensin 2 must be targets as adjuvant for future cancer immunotherapeutic agent.

Genomic evaluation of native Walleye in the Appalachian region and the effects of stocking

AbstractObjectiveCreate a quicker and more accurate genetic assignment tool for Walleye Sander vitreus broodstock in the Eastern Highlands region and to quantify genetic diversity of four Walleye populations using next‐generation sequencing.MethodsTo determine the impacts of stocking nonnative Great Lakes strain Walleye on local populations, three Appalachian populations were sampled: two native populations (Rockcastle River, Kentucky, and New River, Virginia) and one population founded from the Great Lakes strain (Tygart Lake, West Virginia). Walleye from Lake Erie were used as a reference for the Great Lakes strain. Utilization of a genotype‐by‐sequencing approach supported genome‐wide estimates of genetic diversity, population structure, and creation of two single‐nucleotide polymorphism assays that can be used to rapidly identify Great Lakes‐strain, native Eastern Highland‐strain, and F1 hybrid Walleye.ResultResults indicate that the four populations we evaluated were genetically distinct from one another and that each population contains varying degrees of genetic differentiation relative to its source population. The stocked Tygart Lake population displayed lower genetic diversity in metrics such as nucleotide diversity (0.172 vs. 0.184), private alleles (4057 vs. 7623), and observed heterozygosity (0.163 vs. 0.204), likely indicative of genetic drift stemming from a founder effect. The two native populations displayed varying levels of genetic diversity. The New River population was found to have a higher ancestry of the Great Lakes strain in their genome than the Rockcastle River population, reflecting the known admixture of New River Walleye following historic stocking of Great Lakes‐derived Walleye. Our results also identified a pure native Eastern Highlands strain population that can be used for future augmentation and restoration of Eastern Highlands Walleye.ConclusionOur results provide a diagnostic single‐nucleotide polymorphism assay to quickly identify Great Lake strain, Eastern Highland strain, and their F1 hybrid for future management efforts and provide key population genetic insights to managers to enhance best management practices.

The role of type I interferon on trisomy 21 endothelial angiogenesis

Rationale: Down syndrome (DS), resulting from Trisomy 21 (T21), is the most common chromosomal disorder worldwide. Cardiopulmonary complications are a leading cause of morbidity and mortality in DS. Pulmonary disorders include pulmonary hypertension and pulmonary capillaritis, which can result from a dysregulated lung endothelium. One of the major pathways consistently activated in DS is the type I interferon (IFN-I) pathway. High IFN-I inhibits VEGF induced development of capillary like structures, thus playing an anti-angiogenic role. Therefore, we aim to characterize the effects of IFN-I on T21 and euploid iPSC-derived endothelial cells (iPSC-ECs). Methods: Endothelial cell populations from single cell sequencing data of age matched T21 and Non-T21 prenatal lungs were carefully analyzed. Four iPSC lines were derived from age and sex matched T21 and non-T21 human fetal lung fibroblasts and used for endothelial cell differentiation using standardized kits and protocols from Stem Cell Technologies. FACs analysis confirmed endothelial cell purity and differentiation success: CD31+CD34+CD144+CD45-. For angiogenesis assays, endothelial cells were plated onto Cultrex in Ibidi angiogenesis 96-well plates with varying concentrations (0 to 10 4 pM) of IFNβ for 24 hours. Phase contrast images were taken with a 2.5X lens and analyzed in FIJI with the polygon shape tool to measure continuous mesh areas (CMA), defined here as a continuous endothelial cell layer undisrupted by branching structures. Results: Pathway analysis from our scRNAseq data demonstrated an upregulation of the IFN-I pathway across T21 endothelial clusters. T21 and non-T21 iPSC lines exhibited markers of pluripotency (OCT4, NANOG, SOX2, LIN28) and successfully generated pure populations of endothelial cells (~98% CD31+CD34+CD144+CD45-) by passage 2. Quantification demonstrated that at baseline T21 iPSC-ECs consistently had a significantly larger mean CMA than that of non-T21 iPSC-ECs (p< 0.01, n=4). High concentrations of IFN treatment (10 4 pM) resulted in an increased CMA in non-T21 cultures (p=0.14; n=4), trending towards baseline T21 cultures (p=0.28, n=4). Additionally, we observed a significant increase in the CMA of T21 cultures treated with 10 4 pM IFNβ (p=0.04, n=4). Conclusion: Our data suggest decreased angiogenesis in T21 iPSC-ECs may be caused by hyperactive IFN-I signaling. Further studies are needed to determine the mechanisms by which IFN-I regulates angiogenesis in T21 lungs. NIH/NHLBI/OD HL155104-01A1, NIH/NHLBI HL141856, NIH/NHLBI R21HL165411-01, NIH/NICHD 2R24HD000836-52 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mammalian Chromosome Analysis and Sorting by Flow Cytometry.

The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single-chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mitotic block and cell harvesting Basic Protocol 2: Propidium iodide isolation Support Protocol 1: Swelling test Basic Protocol 3: MgSO4 low-molecular-weight isolation Basic Protocol 4: Polyamine high-molecular-weight isolation Support Protocol 2: Molecular-weight determination of chromosomal DNA Basic Protocol 5: Chromosome analysis and sorting.

Human milk extracellular vesicles protect protein cargo from human digestion and are taken up by neonatal human enteroids

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. For example, HM is the best-known preventative for the deadly neonatal intestinal inflammatory disease, necrotizing enterocolitis (NEC), for which there is no cure. How HM reduces NEC risk is relatively unknown. HM contains thousands of molecular components, including extracellular vesicles (EVs). EVs are lipid bilayer-encased particles released from cells that carry biological cargo. EVs are putative regulators of intestinal function and HM EVs offer a mechanism for the transfer of proteins from the mom to the infant’s gut. It is unknown if milk EVs or protein cargo survive digestion. This limits the ability to leverage HM EV proteins as treatments for NEC, additives to infant nutrition, or therapeutics for other diseases. The objective of this proposal is to examine if HM EV protein cargo survives in vivo human digestion and if surviving cargo confers anti-inflammatory benefits in neonatal human enteroids. Hypothesis: HM EVs transport key protein cargo to intercellular targets and protect against intestinal inflammation. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from naso- or orojejunal sampling tubes. EVs were isolated from HM and digesta by density-gradient ultracentrifugation. EVs were validated by electron microscopy, nanoparticle tracking analysis, and western blot. EV protein cargo from n=3 paired HM and digesta samples were profiled by a C18-UPLC with Orbitrap mass spectrometry. Apical-out neonatal human enteroids were validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid EV uptake was assessed with CMPTX dye-labeled EVs. Effects of digesta EVs on inflammation were assessed by qPCR in LPS-treated enteroids. Results: EVs are enriched for markers CD63, CD81, CD9, and TSG101 and de-enriched for casein, milk fat globules. Only 4.68% ± 0.02 (p=0.0012 vs HM) of EV cargo proteins from HM reach the human intestine, but nearly half 64.08% ± 0.02 (p=0.007 vs HM) of the protein diversity is preserved. Human mammary-derived protein BTN1A1 is present in digesta EVs. Apical-out enteroids gene expression is consistent with a more differentiated epithelium, e.g. elevated ChgA (7.60 ± 4.04 vs 1.37 ± 0.39, p=0.0026) and down-regulated Ki67 (0.13 ± 0.04 vs 1.00 ± 0.04, p<0.0001) Enteroids take up milk EVs. Filtered (0.22μm) neonatal digesta (contains EVs) attenuates LPS-induced TNFα gene expression. Conclusion: These novel findings demonstrate that a mostly pure population of EVs can be isolated from a 1mL starting volume. A subset of HM EVs reaches the neonatal human intestine where they can be taken up by the epithelium and may act to reduce inflammation. These data demonstrate the importance of examining EV cargo that survives to the human intestine when investigating potential HM-mediated mechanisms of disease prevention. NIH K01DK129401, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

First Report of the Root-Knot Nematode Meloidogyne enterolobii Parasitizing Eggplant in Mexico.

Eggplant (Solanum melongena L.) is an important vegetable cultivated in Mexico and the state of Sinaloa is the largest producer of eggplants with 90% of the country's total production. In April 2022, eggplants cv. Barcelona exhibiting root-knot, stunted growth, and yellowing were detected in a greenhouse in Culiacán, Sinaloa, Mexico. Disease incidence was approximately 10% (1000 plants evaluated). Ten soil samples were collected from the greenhouse. An average of 400 root-knot nematode second-stage juveniles (J2s) were extracted from 100 g of soil for each sample. Roots were washed with tap water and dissected. Females and egg masses were obtained by dissecting galls. Microscopic examination of the perineal pattern of mature females (n= 10) was round to ovoid, with rounded and high dorsal arch. Females (n= 20) were globular to pear-shaped, body length of 645 to 739 µm, body width of 470 to 559 μm; the stylet was dorsally curved, 15.1 to 16.2 μm long, and with rounded stylet knobs; neck length of 195 to 202 µm and the distance from the base of the stylet to the dorsal gland orifice (DGO) was 4.2 to 5.8 µm. Second-stage juveniles were vermiform, annulated, and tapering at both ends. Morphological characteristics of the females and J2s were consistent with those reported for Meloidogyne enterolobii (Yang and Eisenback 1983). For molecular identification, total DNA was extracted from individual females according to the extraction protocol described by Hu et al. (2011), and the ribosomal intergenic spacer 2 (IGS2) was amplified by PCR using the specific primers Me-F/Me-R for M. enterolobii (Long et al. 2006). PCR amplification generated a 236-bp fragment for the analyzed sample and the amplicon was sequenced. The sequence was deposited in GenBank under the accession number OP004802. BLASTn searches showed 100% identity with available sequences of M. enterolobii from the USA (MH800967) and China (KP411228, MT742011). A phylogenetic tree including published IGS2 sequences for Meloidogyne spp. was constructed based on Maximum Likelihood method. The phylogenetic analysis placed the sequence MeCUB in the same clade with Meloidogyne enterolobii. Pathogenicity tests were performed under greenhouse conditions by inoculating 5000 eggs of a pure population of M. enterolobii on 10 healthy eggplants cv. Barcelona (30-day-old) grown in pots with sterilized soil. Five uninoculated eggplants were used as control. Plants were maintained at 26 to 34°C in a greenhouse for 35 days. Stunted growth and root-galling symptoms appeared on inoculated plants after 21 days, whereas control plants remained symptomless. Nematode reproduction factor (final population density/initial population density) was 0.93 and 2.28 at 28 and 35 days after inoculation, respectively. The nematode on the inoculated roots was morphologically identical to that observed on naturally infected roots in the field. The pathogenicity test was carried out twice with similar results. Meloidogyne enterolobii has been previously reported on eggplants in Puerto Rico (Rammah and Hirschmann 1988). To our knowledge, this is the first report of M. enterolobii causing root-knot of eggplant in Mexico. This nematode is widely distributed in Sinaloa affecting other vegetable crops such as tomato (Martínez-Gallardo et al. 2015), chili (Carrillo-Fasio et al. 2020), and cucumber (Gómez-González et al. 2020), so future studies are required to evaluate integrated management strategies.

Efficient Isolation and Expansion of Limbal Melanocytes for Tissue Engineering.

Limbal melanocytes (LMs) are found in the corneoscleral limbus basal epithelial layer and interact with neighboring limbal epithelial progenitor cells. The difficulty of isolating and cultivating LMs is due to the small fraction of LMs in the overall limbal population and the frequent contamination of primary cultures by other cell types. This has limited the research on freshly isolated LMs and the investigation of their biological significance in the maintenance of the limbal stem cell niche. Here, we describe an optimized protocol for the efficient isolation and expansion of LMs from cadaveric corneal limbal tissue using CD90 and CD117 as selective markers in fluorescence-activated cell sorting to obtain a pure population of LMs (CD90- CD117+) with self-renewal capacity and sustained melanin production. The isolation of pure LMs from a single preparation enables direct transcriptomic and proteomic analyses, as well as functional studies on freshly isolated LMs, which can be considered the proper counterparts of LMs in vivo and have potential applications in tissue engineering.

Genetic dissection, relationship and population structure of drumstick (Moringa oleifera Lam.) using Agro-morphological and SCoT markers

Drumstick (Moringa oleifera Lam.) is an economically important multipurpose perennial vegetable and every part of this plant contain essential properties for nutritional and medicinal values. Specially its leave can be used as an ideal remedy to combat micronutrient-related malnutrition. However, the assessment of genetic diversity is necessitated to isolate rich nutrient dense genotypes for improving culinary, pharmaceutical, and industrial demands. Therefore, In the present study, a total of 52 accessions collected from diversified areas of southern (Tamil Nadu, Kerala, Karnataka, and Andhra Pradesh); western (Maharashtra, Gujarat), and eastern state (Orissa) of India were assessed for morpho-molecular genetic diversity using 10 morphological traits and 36 Start Codon Targeted (SCoT) markers. Out of 36 SCoT primers, 20 primers were polymorphic, and the polymorphic information content (PIC) value ranged from (0.01–0.36) with mean PIC value of 0.21. The neighbor-joining (N-J) tree grouped the 52 accessions into three major distinct clusters (CI-CIII) and the population structure analysis divided these accessions into two pure population groups (probability score ≥ 0.80). Molecular variance using principal component analysis (PCA) showed 30.25% variations in the first three axes (axis 1–13.33%, axis 2–9.33%, and axis 3–7.59%, respectively). Analysis of molecular variance (AMOVA) indicated that 17% variance observed among the population and the remaining 83% variance attributed to variation within individuals. The present investigation elucidates a high genetic diversity among the drumstick accessions, and some distinct accessions could be utilized for crop improvement programmes in trait-specific breeding.

Abstract 1292: CX3CR1 surface expression and phenotypic plasticity in metastasis-initiating cells

Abstract Metastasis-initiating cells (MICs) display stem cell-like features and cause metastatic recurrences leading to patients' demise. We found that prostate and breast cancer cells with high surface expression of the chemokine receptor CX3CR1 (CX3CR1high) account for ~5-7% of the whole population and are endowed with metastasis-initiating ability and exhibits stemness features, including expression of pluripotency genes Nanog and Oct4a. Notably, impairing CX3CR1 signaling hinders metastatic initiation and progression in mouse models of human disease. We also found that cancer cells lacking surface CX3CR1 expression (CX3CR1low) can re-acquire CX3CR1-associated features over time by undergoing phenotypic plasticity. Accordingly, when grafted as pure CX3CR1low populations these cells could generate disseminated tumors in mice, implying that MICs can emerge from cancer cells lacking stemness features. In vitro studies by flow cytometry show that cell surface re-exposure of CX3CR1 by CX3CR1low cells begins within 2 days post-sorting, with the whole population reaching phenotypic equilibrium at 12-16 days post-sorting. Further mechanistic studies revealed that both CX3CR1 protein trafficking and transcriptional regulation play a role in phenotypic plasticity. Additionally, disseminated tumors in mice harbor higher percentages of CX3CR1high cells than in vitro cultures, suggesting that the tissue microenvironment of target organs promotes phenotypic plasticity. These findings nominate CX3CR1 as a novel marker of MICs and provide conceptual ground for future development of approaches targeting CX3CR1 signaling and (re)expression as therapeutic means to prevent or contain metastasis initiation and progression. Citation Format: Jieyi Zhang, Yetunde Oyende, Jennifer Lynch, Olimpia Meucci, Alessandro Fatatis. CX3CR1 surface expression and phenotypic plasticity in metastasis-initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1292.

Abstract 1707: Establishment of a targeted exosome drug delivery system containing natural bacterial toxins and antitoxins

Abstract Background: The inactivation of p53, a tumor suppressor, and the activation of the Ras oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin-antitoxin (TA) system, controlled by Ras and p53, delivered via adenoviruses can be used to selectively and effectively eradicate Ras mutated cancer cells (Shapira et al., oncogene, 2021). Because of their low immunogenicity and known advantage as ideal cell transporters, exosomes offer great promise as cancer immunotherapy agents. CD24 is a GPI-anchored protein that highly expressed in most of human malignancies and not normal cells. Aim: Development of a drug delivery platform for effective and selective eradication of cancer cells while sparing normal cells based on the Trojan horse strategy and genetic signature. Methods: Two delivery systems have been designed and developed; targeted lentivirus and exosomes. Trex293 cells were used to establish stable scFv-CD24-expressing clones, which were adapted to suspension culture in a chemically-defined media. Exosomes were purified by polymer precipitation method and characterized by several methods including nano-particle tracking analysis, Western blot and EXO-ELISA. CD24 stable transfected HCT116-/- (P53 null) and HCT116+/+ (WTP53) clones were established, and evaluated for CD24 and P53 expression. HCT116+/+-CD24 cells were treated with exosomes loaded with GFP or MazF expressing plasmids and analyzed for GFP expression and cell viability. Results: Targeted lentiviruses carrying the TA system effiently shrinked CRC-derived xenografts tumors in mice. Then, a new genetically encoded pseudotyping platform, based on exosomes, was developed. A homogenic and pure population of exosomes was purified from 3D culture of the highest expressing clone (TREx-scFv). Stable and high expression of CD24 were shown, by Western blot and flow cytometry, in both, HCT116-/- and HCT116+/+ cells. HCT116+/+-CD24 cells treated with 5FU showed active P53. The ability of loading DNA into the purified exosomes and deliver them to the target cells were evaluated and showed high and intensive GFP expression. mazF was successfully loaded into the scFv targeted-exosomes and induced a massive cell death, in a dose-dependent manner. Conclusions: By exploiting the P53 genetic status as well as the Ras pathway, an effective and safe treatment could be developed that targets tumor cells specifically while sparing normal tissues. The effectiveness of this out-of-the-box approach holds great promise for the treatment and management of cancer. Citation Format: Shiran Shapira, Daniela Eisurovich, Dina Kazanov, Nadir Arber. Establishment of a targeted exosome drug delivery system containing natural bacterial toxins and antitoxins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1707.

Primary and Immortalized Cultures of Human Proximal Tubule Cells Possess Both Progenitor and Non-Progenitor Cells That Can Impact Experimental Results.

Studies have reported the presence of renal proximal tubule specific progenitor cells which co-express PROM1 and CD24 markers on the cell surface. The RPTEC/TERT cell line is a telomerase-immortalized proximal tubule cell line that expresses two populations of cells, one co-expressing PROM1 and CD24 and another expressing only CD24, identical to primary cultures of human proximal tubule cells (HPT). The RPTEC/TERT cell line was used by the authors to generate two new cell lines, HRTPT co-expressing PROM1 and CD24 and HREC24T expressing only CD24. The HRTPT cell line has been shown to express properties expected of renal progenitor cells while HREC24T expresses none of these properties. The HPT cells were used in a previous study to determine the effects of elevated glucose concentrations on global gene expression. This study showed the alteration of expression of lysosomal and mTOR associated genes. In the present study, this gene set was used to determine if pure populations of cells expressing both PROM1 and CD24 had different patterns of expression than those expressing only CD24 when exposed to elevated glucose concentrations. In addition, experiments were performed to determine whether cross-talk might occur between the two cell lines based on their expression of PROM1 and CD24. It was shown that the expression of the mTOR and lysosomal genes was altered in expression between the HRTPT and HREC24T cell lines based on their PROM1 and CD24 expression. Using metallothionein (MT) expression as a marker demonstrated that both cell lines produced condition media that could alter the expression of the MT genes. It was also determined that PROM1 and CD24 co-expression was limited in renal cell carcinoma (RCC) cell lines.

Generating Functional and Highly Proliferative Melanocytes Derived from Human Pluripotent Stem Cells: A Promising Tool for Biotherapeutic Approaches to Treat Skin Pigmentation Disorders

Melanocytes are essential for skin homeostasis and protection, and their loss or misfunction leads to a wide spectrum of diseases. Cell therapy utilizing autologous melanocytes has been used for years as an adjunct treatment for hypopigmentary disorders such as vitiligo. However, these approaches are hindered by the poor proliferative capacity of melanocytes obtained from skin biopsies. Recent advances in the field of human pluripotent stem cells have fueled the prospect of generating melanocytes. Here, we have developed a well-characterized method to produce a pure and homogenous population of functional and proliferative melanocytes. The genetic stability and potential transformation of melanocytes from pluripotent stem cells have been evaluated over time during the in vitro culture process. Thanks to transcriptomic analysis, the molecular signatures all along the differentiation protocol have been characterized, providing a solid basis for standardizing the protocol. Altogether, our results promise meaningful, broadly applicable, and longer-lasting advances for pigmentation disorders and open perspectives for innovative biotherapies for pigment disorders.

Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device

BackgroundSequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications.ResultsHere, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies’ MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing.ConclusionsOur pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

Pluripotent stem cells: Basic biology or else differentiations aimed at translational research and the role of flow cytometry.

Pluripotent stem cell research has revolutionized the modern era for the past 14 years with the advent of induced pluripotent stem cells. Before this time, scientists had access to human and mouse embryonic stem cells primarily for basic research and an attempt towards lineage-specific differentiations for cell therapy applications. Regarding pluripotent stem cells, expression of bonafide marker proteins such as Oct4, Nanog, Sox2, Klf4, c-Myc, and Lin28 have been considered giving a perfect readout for pluripotent stem cells and assessed using an analytical flow cytometer. In addition to the intracellular markers, surface markers such as stage-specific embryonic antigen-1 for mouse cells and SSEA-4 for human cells are needed to sort pure populations of stem cells for further downstream applications for cell therapy. The surface marker SSEA-4 is the most appropriate for obtaining pure populations of human pluripotent stem cells. When differentiated in a controlled manner using growth factors or small molecules, it is mandatory to assess the downregulation of pluripotency markers (Oct4, Nanog, Sox2, and Klf4) with subsequent up-regulation of stage-specific differentiation markers. Such assessments are done using flow cytometry. Pluripotent stem cells have a high teratoma-forming potential in vivo. Small amounts of undifferentiated PSCs might lead to dangerous teratomas upon transplantation if leftover in the pool of differentiated cells. Hence, flow cytometry is essential for sorting out PSC populations with teratoma-forming potential. The pure populations of differentiated progenitors need to be flow-sorted before differentiating them further for cell therapy applications. For example, Glycoprotein 2 is a specific cell-surface marker for pancreatic progenitors that enables one to sort the pancreatic progenitors differentiated from human PSCs. Taken together, analytical flow cytometry, and cell sorting provide indispensable tools in PSC research and cell therapy.

Abstract P2-11-22: Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes

Abstract Background: Milk whey protein consumption has been associated with breast cancer prevention, therapy and treatment tolerance, and other studies detected these proteins or their mRNAs in breast cancer models or tissues. Our objectives are to ascertain expression of certain whey protein genes in human breast carcinoma cells isolated by Laser Capture Microdissection (LCM) and to determine if gene expression is related to risk of recurrence and overall survival in lesions with various ER/PR status. Genes selected were: LALBA (alpha-lactalbumin), LPO (lactoperoxidase), LTF (lactotransferrin), PAEP (progestogen associated endometrial protein or glycodelin) and SSP1 (secreted phosphoprotein 1 or osteopontin). This retrospective investigation is unique in that highly quantified ER and PR protein levels and ESR1 and PGR relative expression from pure populations of carcinoma cells were employed to stratify cancers into ER/PR subcategories to assess relationships of expression of candidate genes with clinical outcomes. Methods: This investigation of primary breast carcinomas from 170 postmenopausal and 77 premenopausal patients used a matchless deidentified dataset of quantified estrogen receptor (ER) and progestin receptor (PR) protein results with clinical follow-up. ER/PR protein levels, expressed as fmol/mg cytosol protein, were quantified earlier from each breast carcinoma with FDA-approved kits, either Radioligand-Binding Assay (LBA, NEN/DuPont) and/or enzyme immunoassay (EIA, Abbott Labs). Patients were treated with the standard of care at the time of diagnosis. The PixCell IleTM (ThermoFisher, Arcturus) LCM instrument was used to procure only carcinoma cells from tissue sections of de-identified, frozen biopsies, using protocols we employed earlier. Total RNA of each fixed specimen was extracted, purified and amplified to obtain relative expression levels of approximately 22,000 genes by microarray, providing an assessment of mRNA in carcinoma cells only. Relationships of relative gene expression, quantified ER/PR and other features of primary breast cancers were analyzed with clinical results using R software v4.2.0 (Vigorous Calisthenics) by Univariable Cox Regression, Kaplan Meier plots and various tests (i.e., Wilcoxon, Log Ranked, Spearman and/or Chi Square tests). Bidirectional elimination stepwise regression was also used to derive best predictors of patient outcomes. Results: Violin plots of expression distribution of each milk protein gene of 247 biopsies regardless of patient menopausal status revealed that LALBA and SSP1 were highly elevated (p < 0.001) in ER+ (n = 146) or PR+ (n = 146) breast cancer cells while PAEP was quite diminished (p < 0.001). No difference was observed in LPO expression in ER+ or PR+ cells and LTF was only elevated slightly (p < 0.05) in ER+ cells. Regardless of ER/PR status of breast cancer, LTF, LALBA, SSP1, AR and ESR1 gene expression levels were increased significantly in postmenopausal versus premenopausal patients while PAEP was decreased (p < 0.001). No difference was observed in LPO or PGR expression. Correlation matrices examined gene to gene and receptor protein to gene relationships. Using Kaplan Meier analyses of each gene alone with Log Ranked Tests of outcomes, only SSP1 expression was associated with clinical outcome, i.e., overexpression was associated with lower PFS and OS (p = < 0.05). Conclusions: Application of bidirectional elimination stepwise regression of gene expression of the five milk proteins collectively with ER/PR protein status of each carcinoma revealed that LALBA gene expression enhanced prediction of PFS in ER+ cancer while PAEP expression was associated with improved prediction of OS in ER+ carcinomas. We report that expression of LALBA and PAEP genes, largely associated with milk production in normal breast tissue, are also expressed in certain breast carcinomas and influence prediction of patient survival. Citation Format: James L. Wittliff, Michael W. Daniels. Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-22.

Computational comparative analysis identifies potential stemness-related markers for mesenchymal stromal/stem cells.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells that reside in multiple tissues are capable of self-renewal and differentiation into various cell types. These properties make them promising candidates for regenerative therapies. MSC identification is critical in yielding pure populations for successful therapeutic applications; however, the criteria for MSC identification proposed by the International Society for Cellular Therapy (ISCT) are inconsistent across different tissue sources. This study aimed to identify potential markers to be used together with the ISCT criteria to provide a more accurate means of MSC identification. Thus, we carried out a computational comparative analysis of the gene expression in human and mouse MSCs derived from multiple tissues to identify the differentially expressed genes that are shared between the two species. We show that six members of the proteasome degradation system are similarly expressed across MSCs derived from bone marrow, adipose tissue, amnion, and umbilical cord. Additionally, with the help of predictive models, we found that the expression profile of these genes correctly validated the identity of the MSCs across all the tissue sources tested. Moreover, using genetic interaction networks, we showed a possible link between these genes and antioxidant enzymes in the MSC antioxidant defense system, thereby pointing to their potential role in prolonging the life span of MSCs. According to our findings, members of the proteasome degradation system may serve as stemness-related markers.

Enrichment of human embryonic stem cell-derived V3 interneurons using an Nkx2-2 gene-specific reporter

V3 spinal interneurons are a key element of the spinal circuits, which control motor function. However, to date, there are no effective ways of deriving a pure V3 population from human pluripotent stem cells. Here, we report a method for differentiation and isolation of spinal V3 interneurons, combining extrinsic factor-mediated differentiation and magnetic activated cell sorting. We found that differentiation of V3 progenitors can be enhanced with a higher concentration of Sonic Hedgehog agonist, as well as culturing cells in 3D format. To enable V3 progenitor purification from mixed differentiation cultures, we developed a transgene reporter, with a part of the regulatory region of V3-specific gene Nkx2-2 driving the expression of a membrane marker CD14. We found that in human cells, NKX2-2 initially exhibited co-labelling with motor neuron progenitor marker, but V3 specificity emerged as the differentiation culture progressed. At these later differentiation timepoints, we were able to enrich V3 progenitors labelled with CD14 to ~ 95% purity, and mature them to postmitotic V3 interneurons. This purification tool for V3 interneurons will be useful for in vitro disease modeling, studies of normal human neural development and potential cell therapies for disorders of the spinal cord.

The racist origins, racialist connotations, and purity assumptions of the concept of "admixture" in human evolutionary genetics.

The concept of admixture is currently widely being used, both in population genetics research and in DNA ancestry testing discourse. It is assumed to describe the process of gene flow between 2 previously distinct populations that eventually become admixed because of this flow. The concept per se does not require pure or unadmixed populations; the changes are relative and what matters is the level of admixture before and after the event under consideration. However, in this paper, we argue that the concept of admixture as currently used assumes the existence of pure or unadmixed categories. These do not need to have actually existed but to be able to exist in principle. We argue that this is a problematic notion that accrues from the racialist origins of the term admixture, which, as a result, is based on assumptions about purity. We suggest that scientists should be very cautious in their use of this term, especially in science education and communication. We also suggest that the term admixture should be better replaced by terms denoting similarity rather than difference.