Gene Expression In Stem Cells Research Articles

Effect of low dose honey on the apoptosis and inflammation gene expression in corneal limbal stem cells and keratocytes and its efficacy as an ophthalmic formulation in the treatment of dry eye: in-vitro and clinical study

BackgroundThe use of honey as an eye treatment encounters challenges due to its high osmolarity, low pH, and difficulties in sterilization. This study addresses these issues by employing a low concentration of honey, focusing on both in-vitro experiments and clinical trials for treating dry eye disease in corneal cells.MethodsIn the in-vitro experiment, we investigated the impact of a 1% honey-supplemented medium (HSM) on limbal stem cells (LSCs) and keratocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and real-time polymerase chain reaction (PCR) for BCL-2, BAX, and IL-1β gene expression. Simultaneously, in the clinical trial, 80 participants were divided into two groups, receiving either a 1% w/v honey ophthalmic formulation or a placebo for 3 months. Study outcomes included subjective improvement in dry eye symptoms, tear break-up time (TBUT), and Schirmer’s test results.ResultsMTT results indicated that 1% HSM did not compromise the survival of corneal cells and significantly reduced the expression of the IL-1β gene. Additionally, participants in the honey group demonstrated a higher rate of improvement in dry eye symptoms and a significant enhancement in TBUT values at the three-month follow-up. However, there was no significant difference between the study groups in terms of Schirmer’s test values. No adverse events were observed or reported.ConclusionIn conclusion, 1% honey exhibits anti-inflammatory and anti-infective properties, proving effective in ameliorating dry eye symptoms and enhancing tear film stability in patients with dry eye disease.Clinical Trial Registration: https://irct.behdasht.gov.ir/trial/63800.

Differences in the response of normal oral mucosa, oral leukoplakia, oral squamous cell carcinoma-derived mesenchymal stem cells, and epithelial cells to photodynamic therapy

ObjectiveThe objective of this study is to investigate the variances in transcriptome gene expression of normal oral mucosa-derived mesenchymal stem cell (OM-MSC), oral leukoplakia-derived MSC (OLK-MSC) and oral squamous cell carcinoma-derived MSC(OSCC-MSC). as Additionally, the study aims to compare the in vitro proliferation, migration, invasion ability, and response to photodynamic therapy (PDT) of these three MSC, HOK, DOK, leuk1, and Cal27 cell lines. MethodsHOK, DOK, leuk1, Cal27 cells were cultured in vitro. 3 MSC cells were obtained from OM, OLK, OSCC tissue (n = 3) and identified through flow cytometry. They were also cultured in vitro for osteogenic and lipogenic-induced differentiation. Based on the Illumina HiSeq high-throughput sequencing platform, OM-MSC, OLK-MSC, OSCC-MSC (n = 3) were subjected to transcriptome sequencing, functional annotation, and enrichment analysis of differentially expressed genes and related genes. CCK8 assay, wound healing assay, and transwell assay were performed to compare the proliferation, migration, and invasion of the seven types of cells. The 7 cells were incubated with 0, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM of the photosensitizer (5-aminolevulinic acid, 5-ALA) in vitro. Subsequently, they were irradiated with a 150 mM, 635 nm laser for 1 min, and the cell activity was detected using the CCK8 assay after 24 h. The mitochondrial changes in the 7 cells before and after the treatment of PDT were detected using the JC-10 probe, and the changes in ATP content were measured before and after the PDT treatment. ResultsOM-MSC, OLK-MSC, and OSCC-MSC expressed positive MSC surface markers. After osteogenic and lipogenic-induced differentiation culture, stained calcium nodules and lipid droplets were visible, meeting the identification criteria of MSC. Pathway enrichment analysis revealed that the differentially expressed genes (DEGs) of OSCC-MSC compared to OLK-MSC were primarily associated with the PI3K-Akt signaling pathway and tumor-related pathways. OSCC-MSC exhibited stronger migratory and invasive abilities compared to Cal27. The IC50 values required for OM, OLK, and OSCC-derived MSC were lower than those required for epithelial cells treated with PDT, which were 1.396 mM, 0.9063 mM, and 2.924 mM, respectively. Cell membrane and mitochondrial disruption were observed in seven types of cells after 24 h of PDT treatment. However, HOK, DOK, leuk1, and Cal27 cells had an ATP content increased. ConclusionsOLK, OSCC epithelial cells require higher concentrations of 5-ALA for PDT treatment than MSC of the same tissue origin. The concentration of 5-ALA required increases with increasing cell malignancy. Differences in the response of epithelial cells and MSC to PDT treatment may have varying impacts on OLK recurrence and malignancy.

Identification of Drug Targets and Agents Associated with Ferroptosis-related Osteoporosis Through Integrated Network Pharmacology and Molecular Docking Technology.

Osteoporosis is a systemic bone disease characterized by progressive reduction of bone mineral density and degradation of trabecular bone microstructure. Iron metabolism plays an important role in bone; its imbalance leads to abnormal lipid oxidation in cells, hence ferroptosis. In osteoporosis, however, the exact mechanism of ferroptosis has not been fully elucidated. The main objective of this project was to identify potential drug target proteins and agents for the treatment of ferroptosis-related osteoporosis. In the current study, we investigated the differences in gene expression of bone marrow mesenchymal stem cells between osteoporosis patients and normal individuals using bioinformatics methods to obtain ferroptosis-related genes. We could predict their protein structure based on the artificial intelligence database of AlphaFold, and their target drugs and binding sites with the network pharmacology and molecular docking technology. We identified five genes that were highly associated with osteoporosis, such as TP53, EGFR, TGFB1, SOX2 and MAPK14, which, we believe, can be taken as the potential markers and targets for the diagnosis and treatment of osteoporosis. Furthermore, we observed that these five genes were highly targeted by resveratrol to exert a therapeutic effect on ferroptosis-related osteoporosis. We examined the relationship between ferroptosis and osteoporosis based on bioinformatics and network pharmacology, presenting a promising direction to the pursuit of the exact molecular mechanism of osteoporosis so that a new target can be discovered for the treatment of osteoporosis.

In vitro aging alters the gene expression and secretome composition of canine adipose-derived mesenchymal stem cells.

Canine adipose-derived mesenchymal stem cells (cAD-MSCs) hold therapeutic promise due to their regenerative potential, particularly within their secretome. However, concerns arise regarding the impact of in vitro cultivation necessitated for storing therapeutic doses, prompting this study to comprehensively explore the impact of in vitro aging on gene expression and secretome composition. The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling using RT-qPCR array, and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during in vitro culture. The results demonstrate that the gene expression and secretome composition of cAD-MSCs were impacted by in vitro aging. Among many alterations in gene expression between two passages, two significant downregulations were noted in the MSC-associated PTPRC and IL10 genes. While the majority of proteins and their functional characteristics were shared between passages, the influence of cell aging on secretome composition is highlighted by 10% of proteins being distinctively expressed in each passage, along with 21 significant up- and downregulations. The functional attributes of proteins detected in passage 3 demonstrated a greater inclination towards supporting the regenerative capacity of cAD-MSCs. Moreover, proteins in passage 6 exhibited a noteworthy correlation with the blood coagulation pathway, suggesting an elevated likelihood of coagulation events. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age in vitro. Furthermore, it contributes to broadening the currently restricted knowledge base concerning the secretome of cAD-MSCs. In conclusion, our findings show that the regenerative potential of cAD-MSCs, as well as their secretome, may be compromised by in vitro aging. Therefore, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.

Abstract 1687: Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring

Abstract Hematopoiesis relies on an intricate balance between stem-cell self-renewal and terminal maturation of blood cells. Disrupting this balance can lead to the development of deadly blood cancers, like acute myeloid leukemia (AML). Increasing evidence suggests that acquiring hematopoietic stem cell (HSC) gene expression programs in AML confers a particularly poor prognosis and increases risk of relapse with conventional therapy. Acquisition of these features in AML is commonly driven by dysregulation of MECOM, a transcription factor and master regulator of HSC self-renewal. To define the role of MECOM in AMLs, we engineered cell-line models of high-risk AML by endogenously tagging MECOM with an FKBP12F36V degron. These models enable targeted degradation of MECOM which we employed in concert with multi-omic readouts to assess changes in nascent transcription and chromatin accessibility to identify direct transcriptional targets of MECOM. We demonstrate that MECOM degradation results in a rapid increase in chromatin accessibility at MECOM-bound sites and increased expression of cognate genes to establish a pro-differentiation gene program. These findings suggest a previously unappreciated mechanism for MECOM in repressing differentiation-promoting cis-regulatory elements (cisREs) and their cognate target genes in AML. We then employed a pooled CRISPR inhibition (CRISPRi) screen to functionally characterize the role that each cisRE plays in promoting high-risk features in AML. Utilizing CRISPRi to repress MECOM-bound cisREs in concert with dTAG-mediated MECOM degradation, we examined if repression of over 500 individual MECOM-bound cisREs is sufficient to maintain AML progenitors in the absence of MECOM. This functional dissection revealed that the individual repression of three cisREs that regulate CEBPA, GFI1B, and RUNX1 is sufficient to maintain AML progenitor cells in the absence of MECOM. This result implicates MECOM as a direct repressor of known regulators of myeloid differentiation. Given these findings, we hypothesized that small molecule-based recruitment of a transcriptional coactivator to MECOM could rewire its repressive function to activate myeloid differentiation and subsequent leukemia cell death. To test this approach, we treated a MECOM-FKBP12F36V AML degron model with the bifunctional small molecule NICE-01 (AP1867-PEG2-JQ1) to recruit an epigenetic activator (BRD4) to MECOM to functionally rewire this transcriptional network. NICE-01 treatment induced significant differentiation of AML progenitor cells relative to JQ1 treatment alone. Our work highlights the utility of targeted protein degradation to mechanistically interrogate the function of a key driver of high-risk AMLs and suggests the potential for small molecule-rewiring of stem cell gene regulatory networks to confer therapeutic benefit. Citation Format: Travis J. Fleming, Michael Gundry, Richard Voit, Mateusz Antoszewski, William J. Gibson, Ananthan Sadagopan, Vijay G. Sankaran. Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1687.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1566.

Cellular abundance-based prognostic model associated with deregulated gene expression of leukemic stem cells in acute myeloid leukemia.

Background: Previous studies have reported that genes highly expressed in leukemic stem cells (LSC) may dictate the survival probability of patients and expression-based cellular deconvolution may be informative in forecasting prognosis. However, whether the prognosis of acute myeloid leukemia (AML) can be predicted using gene expression and deconvoluted cellular abundances is debatable. Methods: Nine different cell-type abundances of a training set composed of the AML samples of 422 patients, were used to build a model for predicting prognosis by least absolute shrinkage and selection operator Cox regression. This model was validated in two different validation sets, TCGA-LAML and Beat AML (n = 179 and 451, respectively). Results: We introduce a new prognosis predicting model for AML called the LSC activity (LSCA) score, which incorporates the abundance of 5cell types, granulocyte-monocyte progenitors, common myeloid progenitors, CD45RA + cells, megakaryocyte-erythrocyte progenitors, and multipotent progenitors. Overall survival probabilities between the high and low LSCA score groups were significantly different in TCGA-LAML and Beat AML cohorts (log-rank p-value = and , respectively). Also, multivariate Cox regression analysis on these two validation sets shows that LSCA score is independent prognostic factor when considering age, sex, and cytogenetic risk (hazard ratio, HR = 2.17; 95% CI 1.40-3.34; p < 0.001 and HR = 1.20; 95% CI 1.02-1.43; p < 0.03, respectively). The performance of the LSCA score was comparable to other prognostic models, LSC17, APS, and CTC scores, as indicated by the area under the curve. Gene set variation analysis with six LSC-related functional gene sets indicated that high and low LSCA scores are associated with upregulated and downregulated genes in LSCs. Conclusion: We have developed a new prognosis prediction scoring system for AML patients, the LSCA score, which uses deconvoluted cell-type abundance only.

Abstract 1812 Vinculin Expression and Substrate Stiffness Regulate Proliferation, Activin-A Secretion and Mechanosensory Gene Expression in Mesenchymal Stem Cells

Abstract 1812 Vinculin Expression and Substrate Stiffness Regulate Proliferation, Activin-A Secretion and Mechanosensory Gene Expression in Mesenchymal Stem Cells

Enhancing Vasculogenesis in Dental Pulp Development: DPSCs-ECs Communication via FN1-ITGA5 Signaling.

Dental pulp regeneration therapy is a challenge to achieve early vascularization during treatment. Studying the regulatory mechanisms of vascular formation during human dental pulp development may provide insights for related therapies. In this study, we utilized single-cell sequencing analysis to compare the gene expression of dental pulp stem cells (DPSCs) and vascular endothelial cells (ECs) from developing and mature dental pulps. Immunohistochemistry, Western blot, and real-time polymerase chain reaction (RT-PCR) were used to detect fibronectin 1 (FN1) expression and molecules, such as PI3K/AKT. Cell proliferation assay, scratch assay, tube formation assay and were used to investigate the effects of DPSCs on the vasculogenetic capability of ECs. Additionally, animal experiments involving mice were conducted. The results revealed that DPSCs exist around dental pulp vasculature. FN1 expression was significantly higher in DPSCs from young permanent pulps than mature pulps, promoting HUVEC proliferation, migration, and tube formation via ITGA5 and the downstream PI3K/AKT signaling pathway. Our data indicate that intercellular communication between DPSCs and ECs mediated by FN1-ITGA5 signaling is crucial for vascularizationduring dental pulp development, laying an experimental foundation for future clinical studies.

Mesenchymal stem cells in PRP and PRF containing poly(3-caprolactone)/gelatin Scaffold: a comparative in-vitro study.

Scaffold design is one of the three most essential parts of tissue engineering. Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) have been used in clinics and regenerative medicine for years. However, the temporal release of their growth factors limits their efficacy in tissue engineering. In the present study, we planned to synthesize nanofibrous scaffolds with the incorporation of PRP and PRF by electrospinning method to evaluate the effect of the release of PRP and PRF growth factors on osteogenic gene expression, calcification, proliferation, and cell adhesion of human bone marrow mesenchymal stem cell (h-BMSC) as they are part of scaffold structures. Therefore, we combined PRP/PRF, derived from the centrifugation of whole blood, with gelatin and Polycaprolactone (PCL) and produced nanofibrous electrospun PCL/Gel/PRP and PCL/Gel/PRF scaffolds. Three groups of scaffolds were fabricated, and h-BMSCs were seeded on them: (1) PCL/Gel; (2) PCL/Gel/PRP; (3) PCL/Gel/PRF. MTS assay was performed to assess cell proliferation and adhesion, and alizarin red staining confirmed the formation of bone minerals during the experiment. The result indicated that PCL/Gel did not have any better outcomes than the PRP and PRF group in any study variants after the first day of the experiment. PCL/gelatin/PRF was more successful regarding cell proliferation and adhesion. Although PCL/gelatin/PRP showed more promising results on the last day of the experiment in mineralization and osteogenic gene expression, except RUNX2, in which the difference with PCL/gelatin/PRF group was not significant.

Mechanisms driving fasting-induced protection from genotoxic injury in the small intestine.

Genotoxic agents such as doxorubicin (DXR) can cause damage to the intestines that can be ameliorated by fasting. How fasting is protective and the optimal timing of fasting and refeeding remain unclear. Here, our analysis of fasting/refeeding-induced global intestinal transcriptional changes revealed metabolic shifts and implicated the cellular energetic hub mechanistic target of rapamycin complex 1 (mTORC1) in protecting from DXR-induced DNA damage. Our analysis of specific transcripts and proteins in intestinal tissue and tissue extracts showed that fasting followed by refeeding at the time of DXR administration reduced damage and caused a spike in mTORC1 activity. However, continued fasting after DXR prevented the mTORC1 spike and damage reduction. Surprisingly, the mTORC1 inhibitor, rapamycin, did not block fasting/refeeding-induced reduction in DNA damage, suggesting that increased mTORC1 is dispensable for protection against the initial DNA damage response. In Ddit4-/- mice [DDIT4 (DNA-damage-inducible transcript 4) functions to regulate mTORC1 activity], fasting reduced DNA damage and increased intestinal crypt viability vs. ad libitum-fed Ddit4-/- mice. Fasted/refed Ddit4-/- mice maintained body weight, with increased crypt proliferation by 5 days post-DXR, whereas ad libitum-fed Ddit4-/- mice continued to lose weight and displayed limited crypt proliferation. Genes encoding epithelial stem cell and DNA repair proteins were elevated in DXR-injured, fasted vs. ad libitum Ddit4-/- intestines. Thus, fasting strongly reduced intestinal damage when normal dynamic regulation of mTORC1 was lost. Overall, the results confirm that fasting protects the intestines against DXR and suggests that fasting works by pleiotropic - including both mTORC1-dependent and independent - mechanisms across the temporally dynamic injury response.NEW & NOTEWORTHY New findings are 1) DNA damage reduction following a 24-h fast depends on the timing of postfast refeeding in relation to chemotherapy initiation; 2) fasting/refeeding-induced upregulation of mTORC1 activity is not required for early (6 h) protection against DXR-induced DNA damage; and 3) fasting increases expression of intestinal stem cell and DNA damage repair genes, even when mTORC1 is dysregulated, highlighting fasting's crucial role in regulating mTORC1-dependent and independent mechanisms in the dynamic recovery process.

Abstract A060: Organoid Culture as a Unique Resource for Studying Aggressive Triple Negative Breast Cancer in African American

Abstract Introduction Triple negative breast cancer (TNBC) among women with African ancestry has been shown to be more aggressive than in women of European ancestry. However, the root cause for this disparity is not fully understood. In our previous studies, we show that the gene expression in the breast cancer stem cells of TNBC tumors was differential based on Western sub-Saharan ancestry. To study the role of African ancestry in the biology of TNBC, we have developed patient-derived organoids (PDO) from African (Ghanaian) and African American tumors that will be available for research. Organoid culture has the capacity to mimic the physiological tissue organization and functionality and has been used as an invaluable tool for disease modeling and drug discovery. Objective The objective is to develop and characterize PDOs based on African TNBC that will serve as a renewable source of African tumors for breast cancer research. Methods Through our international collaboration called Precision Medicine for Aggressive Breast Cancers (PMABC), we obtained fresh patient tumors from continental Africa (Ghana) and African American tumors from Henry Ford Health. PDOs were developed form both primary tumors and patient-derived xenografts (PDX). Tissues were processed using the Miltenyi Gentle Mac. The cells were seeded in a 12-well plate coated with Matrigel. Developed organoids are characterized using single cell RNA sequencing as well as 10X spatial sequencing Results We have successfully developed organoids using frozen tissues from both West African and African American breast patients. We have successfully established four (4) organoid lines from individual primary breast cancer tissues and one PDX. The organoids are passaged after every 7-14 days, and the highest passage so far is passage number 12 for the organoids from primary tissues and passage number 15 for the PDXs. We are in the process of characterizing the developed organoids using single-cell assay in comparisons to their primary tumor of origin. Conclusion Establishment of organoids has helped us have a renewable source of tumor for studying the aggressive TNBC among Women with African ancestry. We aim to use the organoids to identify tumor biomarkers for treating aggressive TNBC that disproportionately affects women of western sub Saharan ancestry. Citation Format: Moses Kamita, Kurt Fernando, Sylvester Antwi, Forster Amponsah-Manu, Josephine Nsaful, Kafui Akakpo, Jessica Bensenhaver, Kwabena Agbedinu, Michael Nortey, Nelson Affram, Mohammed Sheriff, Cassandra Mills, Alex Mremi, Jieoma Aja, Moses Dokurugu, Haythem Ali, Eleanor Walker, Max Wicha, Evelyn Jiagge. Organoid Culture as a Unique Resource for Studying Aggressive Triple Negative Breast Cancer in African American [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A060.

Quantitative association between gene expression and blood cell production of individual hematopoietic stem cells in mice.

Individual hematopoietic stem cells (HSCs) produce different amounts of blood cells upon transplantation. Taking advantage of the intercellular variation, we developed an experimental and bioinformatic approach to evaluating the quantitative association between gene expression and blood cell production across individual HSCs. We found that most genes associated with blood production exhibit the association only at some levels of blood production. By mapping gene expression with blood production, we identified four distinct patterns of their quantitative association. Some genes consistently correlate with blood production over a range of levels or across all levels, and these genes are found to regulate lymphoid but not myeloid production. Other genes exhibit one or more clear peaks of association. Genes with overlapping peaks are found to be coexpressed in other tissues and share similar molecular functions and regulatory motifs. By dissecting intercellular variations, our findings revealed four quantitative association patterns that reflect distinct dose-response molecular mechanisms modulating the blood cell production of HSCs.

Genetically Engineered Probiotic Limosilactobacillus reuteri Releasing IL-22 (LR-IL-22) Modifies the Tumor Microenvironment, Enabling Irradiation in Ovarian Cancer

Despite recent advances in cancer therapy, ovarian cancer remains the most lethal gynecological cancer worldwide, making it crucial and of the utmost importance to establish novel therapeutic strategies. Adjuvant radiotherapy has been assessed historically, but its use was limited by intestinal toxicity. We recently established the role of Limosilactobacillus reuteri in releasing IL-22 (LR-IL-22) as an effective radiation mitigator, and we have now assessed its effect in an ovarian cancer mouse model. We hypothesized that an LR-IL-22 gavage would enable intestinal radioprotection by modifying the tumor microenvironment and, subsequently, improving overall survival in female C57BL/6MUC-1 mice with widespread abdominal syngeneic 2F8cis ovarian cancer. Herein, we report that the LR-IL-22 gavage not only improved overall survival in mice when combined with a PD-L1 inhibitor by inducing differential gene expression in irradiated stem cells but also induced PD-L1 protein expression in ovarian cancer cells and mobilized CD8+ T cells in whole abdomen irradiated mice. The addition of LR-IL-22 to a combined treatment modality with fractionated whole abdomen radiation (WAI) and systemic chemotherapy and immunotherapy regimens can facilitate a safe and effective protocol to reduce tumor burden, increase survival, and improve the quality of life of a locally advanced ovarian cancer patient.

Comparative study of pluripotency-related gene expression in Umbilical Cord Mesenchymal Stem Cells (UC-MSC) from IUGR, SGA and normally grown fetuses

Background and Objectives: Intrauterine growth restriction (IUGR) is worldwide a public health problem. IUGR-born individuals acquire epigenetic “thrifty-phenotype” gene adaptations, according to the “Barker hypothesis” and are more susceptible to develop metabolic related diseases. Small for Gestational Age (SGA) fetuses consist of a transitional group between IUGR and normally grown fetuses (Appropriate for Gestational Age, AGA). Currently, there is limited information in the literature whether IUGR or SGA status affects the pluripotency of the Umbilical Cord Mesenchymal Stem Cell (UC-MSC) with respect to gene expression at various stages of cell culture. Materials and Methods: In this study, we investigated stemness marker expression in UC-MSCs derived from twenty-one AGA, IUGR, and SGA fetuses. The relative expression of key genes involved in stemness, pluripotency, and cell proliferation, namely IGF2BP1, CMYC, GLI1, P21, NANOG, OCT4, and SOX2, was assessed in UC-MSCs at passages P0 and P1 using flow cytometry and quantitative PCR. Results: We found lower median expression levels of IGF2BP1 in IUGR group (P=0.044) and P21 in SGA group (P=0.024) at P0 compared to control group. GLI1 and OCT4 exhibited reduced expression at P1 in the IUGR group (P=0.0434 and P=0.0343 respectively) compared to AGA. NANOG median expression differed statistically between CONTROL and IUGR group in P0 (P=0.034) and P1 (P=0.0205). No significant changes were noted for SOX2 and CMYC median expression among the various groups in P0 and P1. Conclusion: The comparative analysis of stemness marker expression among UC-MSCs from these three different sources is a novel descriptive aspect of our study, adding a new perspective to the existing literature.

Dissolution of ribonucleoprotein condensates by the embryonic stem cell protein L1TD1.

L1TD1 is a cytoplasmic RNA-binding protein specifically expressed in pluripotent stem cells and, unlike its mouse ortholog, is essential for the maintenance of stemness in human cells. Although L1TD1 is the only known protein-coding gene domesticated from a LINE-1 (L1) retroelement, the functional legacy of its ancestral protein, ORF1p of L1, and how it is manifested in L1TD1 are still unknown. Here, we determined RNAs associated with L1TD1 and found that, like ORF1p, L1TD1 binds L1 RNAs and localizes to high-density ribonucleoprotein (RNP) condensates. Unexpectedly, L1TD1 enhanced the translation of a subset of mRNAs enriched in the condensates. L1TD1 depletion promoted the formation of stress granules in embryonic stem cells. In HeLa cells, ectopically expressed L1TD1 facilitated the dissolution of stress granules and granules formed by pathological mutations of TDP-43 and FUS. The glutamate-rich domain and the ORF1-homology domain of L1TD1 facilitated dispersal of the RNPs and induced autophagy, respectively. These results provide insights into how L1TD1 regulates gene expression in pluripotent stem cells. We propose that the ability of L1TD1 to dissolve stress granules may provide novel opportunities for treatment of neurodegenerative diseases caused by disturbed stress granule dynamics.

Gα12 signaling regulates transcriptional and phenotypic responses that promote glioblastoma tumor invasion

In silico interrogation of glioblastoma (GBM) in The Cancer Genome Atlas (TCGA) revealed upregulation of GNA12 (Gα12), encoding the alpha subunit of the heterotrimeric G-protein G12, concomitant with overexpression of multiple G-protein coupled receptors (GPCRs) that signal through Gα12. Glioma stem cell lines from patient-derived xenografts also showed elevated levels of Gα12. Knockdown (KD) of Gα12 was carried out in two different human GBM stem cell (GSC) lines. Tumors generated in vivo by orthotopic injection of Gα12KD GSC cells showed reduced invasiveness, without apparent changes in tumor size or survival relative to control GSC tumor-bearing mice. Transcriptional profiling of GSC-23 cell tumors revealed significant differences between WT and Gα12KD tumors including reduced expression of genes associated with the extracellular matrix, as well as decreased expression of stem cell genes and increased expression of several proneural genes. Thrombospondin-1 (THBS1), one of the genes most repressed by Gα12 knockdown, was shown to be required for Gα12-mediated cell migration in vitro and for in vivo tumor invasion. Chemogenetic activation of GSC-23 cells harboring a Gα12-coupled DREADD also increased THBS1 expression and in vitro invasion. Collectively, our findings implicate Gα12 signaling in regulation of transcriptional reprogramming that promotes invasiveness, highlighting this as a potential signaling node for therapeutic intervention.

Single Cell Analysis Identifies Expression of PD-L2 on Human Bone Marrow Hematopoietic Stem Cells

The identification of specific and reliable markers for human hematopoietic stem cells (HSCs) is crucial to enable their prospective isolation allowing the investigation of unique physiological HSC functions within the blood cell hierarchy. Despite significant advancements in identification of surface markers for human bone marrow HSC isolation, the enriched populations remain heterogeneous. Here we applied single cell CITE-Seq (cellular indexing of transcriptomes and epitopes) of FACS-enriched hematopoietic stem and progenitor cells (HSPCs) to decipher differentiation hierarchies and to identify markers of most immature populations. We FACS-isolated over 60.000 high-quality human bone marrow HSPCs by sorting CD34 + and CD34 +CD38 - cells from 15 different healthy donors. These were subjected to scCITE-Seq using the BD Rhapsody system with a targeted gene panel comprising 600 most relevant genes of early HSC differentiation and 50 antibodies. To validate differentially expressed surface marker genes, we performed extensive in vitro and in vivo studies of FACS-purified subpopulations of human bone marrow and mobilized peripheral blood samples (mPB) from healthy donors. Our multi-omics based single HSPC profiling indicated Programmed death ligand 2 (PD-L2 / CD273) as a surface marker expressed on the most immature HSCs within the hematopoietic compartment defined as CD34 +CD38 -CD45RA -CD90 +. Prospective FACS-based isolation allowed a deeper understanding of transcriptomic differences depending on PD-L2 positivity. A transcriptomic principal component analysis of CD273 hi and CD273 low expressing HSCs isolated pairwise from four healthy donors proved distinct clustering accompanied by discriminative molecular signatures. Stem cell genes as HOPX, MLLT3 and MPL were enriched in CD273 hi HSCs, and CDK6 was downregulated, further validating our single-cell analysis. The differential expression of stem cell genes was validated at protein level. Single cell tracking using video-microscopy confirmed a delayed entry into cell cycle in CD273 hi HSCs, suggesting deeper quiescence of these cells. Furthermore, their mitochondrial potential was reduced. In vivo transplantations of both populations in immunocompromised NSGs are ongoing and will reveal the long-term repopulation potential of CD273 hi HSCs. Multiple in vitro differentiation assays demonstrated CD273 positivity to be associated with an increased stem cell phenotype with higher expression of EPCR (CD201) and CD90 in liquid culture, a delayed differentiation and high capability of generating multipotent colonies. Interestingly, CD273 surface expression was strongly upregulated in HSCs upon in vitro culture. Since PD-L2 is known as a ligand for the Programmed death receptor 1 (PD1), we investigated the immunomodulating function of PD-L2 expressing HSPCs by establishing an allogeneic lymphocyte reaction assay. In addition to their stem cell-like characteristics, PD-L2 expressing HSPCs mediated diminished T cell proliferation in allogeneic co-culture settings. Furthermore, cytokine profiling of co-culture supernatants indicated increased secretion of anti-inflammatory cytokines (IL-10 and TGF-β1), accompanied by reduced secretion of pro-inflammatory cytokines (IL-17A) and a diminished expression of the pro-inflammatory receptor CD69 on CD8 + T cells. These findings showed that CD34 + cells isolated from mobilized peripheral blood of healthy donors possess immunomodulating capabilities, and that PD-L2 expression indicates an immunocompromising HSC phenotype. In conclusion, this study demonstrates differential surface expression of PD-L2 within the most immature human HSCs. PD-L2 expression is associated with HSC quiescence and delayed in vitro differentiation. Furthermore, these results provide valuable insights into the immunomodulating features of HSCs expressing PD-L2.

Single Cell Transcriptomic Analysis of B-Cell Acute Lymphoblastic Leukemia (B-ALL) Reveals Correlation between Leukemia Cell State, Genetic Alterations and Clinical Outcome

I.I., A.G.X.Z, Q.G., L.G.P.: co-first authors; J.E.D., C.G.M.: co-senior authors Introduction: Genomic analyses of bulk ALL samples have improved our understanding of the genetic basis and risk stratification of B-ALL, but do not directly examine intratumor heterogeneity or enable inference of leukemia developmental state and cell of origin. Methods: We profiled 89 B-ALL samples by single-cell RNA-seq (scRNA-seq) (10X Genomics 5'v2) and compared them to a scRNA-seq reference map of normal human B-cell development. Results: Analysis of heterogeneity of inferred DNA copy number alterations at single cell level showed that aneuploid ALL with near haploidy and hyperdiploid harbor chromosomal losses or gains in all blasts, consistent with early, synchronous origin rather than sequential accumulation. A subset of TCF3::PBX1 samples harbored clones with progressive overexpression of genes surrounding PBX1 which scWGS confirmed caused by accumulation of DNA gains. Extensive intra-sample heterogeneity was observed by non-negative matrix factorization and driven by five consensus gene expression signatures: cell cycle (S; G2/M), metabolism, differentiation and inflammation. Signature composition refined leukemic subtyping; for example, within DUX4-r B-ALL two groups were discerned: one with higher inflammation score, expression of stem cell genes and worse outcome and one with lower inflammation score and expression of Pro-B related genes. To understand variation in developmental states among B-ALL blasts, we first developed an atlas of human B cell development. Marker genes elucidated from bulk RNA-seq on purified human HSC, MPP, LMPP, MLP, CLP, Pre-Pro-B, Pro-B, Pre-B, and B populations guided the development of a new scRNA-seq atlas of human B cell development comprising 130,085 cells spanning 90 fetal, pediatric, and adult samples from 8 studies (Fig 1A). Unexpectedly, we found that human CLPs retain transcriptional programs governed by CEBPA; in vitro and in vivo functional assays validated the capacity of CLPs for extensive myeloid differentiation prior to lineage restriction at the Pro-B stage. We next mapped our 89 B-ALL sc samples to precise cellular states along B cell development and clustered samples by leukemia cell composition. As expected, most samples exhibited similarity of GEP to that of pro-B cells, and this was most pronounced among hyperdiploid samples. High pre-B abundance was observed in MEF2D-r and TCF3::PBX1, while an HSC/MPP/LMPP enriched cluster encompassed ZNF384-r and a subset of DUX4-r B-ALL. Notably, one ZNF384-r B-ALL patient with LMPP involvement at diagnosis retained these LMPP-like cells at relapse despite a lineage switch to AML. Finally, we identified a group of patients with high early lymphoid (MLP, CLP, Pre-Pro-B) abundance representing a subset of Ph+ and KMT2A-r samples (Fig. 1B). We next utilized marker genes from each B-ALL cell state to estimate their relative abundance within a bulk RNA-seq cohort of 2046 B-ALL patients. In addition to validating these associations between genomic class and leukemia cell state, we identified age-dependent patterns of B-ALL state involvement wherein early lymphoid involvement is highest in infancy and adulthood while Pro-B involvement is highest in childhood. In childhood, high-risk disease is associated with higher early lymphoid ( P=4.9e-12) and lower Pro-B ( P=0.0085) abundance. Further, patients with residual disease (RD) levels above 1% are enriched for higher upfront early lymphoid abundance ( P=1e-7) and lower Pro-B ( P=4.1e-5) and Pre-B ( P=2.2e-11) abundance compared to patients with RD &amp;lt; 0.01%. Finally, B-ALL cell state improved resolution of previously reported transcriptional heterogeneity within defined genomic classes. Early lymphoid abundance discerns between two subtypes within Ph+ (Kim Nat Gen 2023) with AUC=0.953 ( P=2.4e-9), as well as two subtypes within KMT2A-r (Brady Nat Gen 2022) with AUC=0.996 ( P=1.1e-6). Notably we find that KMT2A::AFF1 fusions produce early lymphoid-enriched disease compared to KMT2A::MLLT3 ( P=0.0024) and KMT2A::MLLT10 ( P=0.0037), wherein the latter two fusions exhibit Pre-B enrichment ( P=0.025 and P=0.0037, respectively). Conclusions: Understanding variation in transcriptional programs and developmental states of B-ALL blasts by sc transcriptome refines existing clinical and genomic classifications and provides novel prognostic markers.

Mutant p53 Drives the Development of Myelodysplastic Syndromes Via Dysregulating Pre-mRNA Splicing in Hematopoietic Stem and Progenitor Cells

Despite the clinical importance of TP53 mutations, to date there have been no studies on the functional impact of TP53 mutations on the initiation and progression of MDS. We found that heterozygous p53 mutant mice ( p53 R248W/+) show extended survival and died within 15-20 months after birth. p53 mutant mice had hypercellular bone marrow and displayed cytopenia, thrombocytopenia, and anemia. Approximately 60% of p53 R248W/+ mice developed MDS based upon pathological analysis of bone marrow (BM) and peripheral blood smears, while the remaining p53 R248W/+ mice developed lymphoma and sarcoma. We further showed that mutant p53-driven MDS are transplantable. Thus, we have generated a mouse model of MDS that has some features of human MDS with TP53 mutations. To understand how mutant p53 drives MDS development, we performed RNA-seq studies to compare gene expression in hematopoietic stem and progenitor cells (HSPCs). GSEA analysis revealed that spliceosome genes were significantly downregulated in middle-aged p53 R248W/+ HSPCs compared to age-matched p53 +/+ HSPCs. We confirmed that the expression of splicing factors, such as Prpf3 and Prpf4, was significantly downregulated in middle-aged p53 R248W/+ HSPCs. We conducted alternative splicing analysis utilizing MISO (Mixture of Isoforms) algorithm to identify differences in exon inclusion ratio between WT and mutant HSPCs. We observed differential splicing of all classes of alternative splicing events, including Cassette Exons (CE), Competing 5′ and 3′ splice sites (A5' or A3'SS), Mutually Exclusive Exons (MXE), and Retained Introns (RI) in p53 R248W/+ cells compared to that of the WT HSPCs. Notably, we found that murine p53 mutant HSPCs exhibited aberrant splicing of key regulators of NFκBactivation, such as USP15. USP15 overexpression promotes NFκB expression through inhibiting its ubiquitination, whereas NFκB promotes USP15 expression. p53 mutant HSPCs tend to skip exon 7 and express high levels of short isoform of Usp15. The short isoform of Usp15, but not the long isoform of Usp15, activates NFκB in HSPCs, manifested by increased phosphorylation of p65. To determine the impact of mutant p53 on pre-mRNA splicing in human HSPCs, we ectopically expressed GFP or p53 R248W in human cord blood CD34 + cells and performed RNA-seq in transduced cells (GFP +). Human HSPCs expressing mutant p53 displayed alterations in pre-mRNA splicing compared to HSPCs expressing GFP. We found that human HSPCs expressing mutant p53 display aberrant splicing in key regulator of NFκB such as IKBKE (Inhibitor of nuclear factor kappa-B kinase subunit epsilon). Human HSPCs expressing mutant p53 tend to include exon 7 and express high levels of long isoform of IKBKE. The long isoform of IKBKE, but not the short isoform of IKBKE, activates NFκB in human HSPCs. Many proinflammatory cytokine genes are NFκB targets and we observed increased levels of pro-inflammatory cytokines, including IL-1β and IL-6, in the BM of middle-aged p53 mutant mice. Notably, we observed increased levels of IL-1β and IL-6 in the BM of MDS or AML patients with TP53 mutations. TP53 mutations co-occur with splicing factor mutations in MDS. To determine whether TP53 and SRSF2 mutations cooperate in pathogenesis of MDS, we have generated p53 R248W/+SRSF2 P95H/+ mice. We found that the survival of recipient mice repopulated with p53 R248W/+SRSF2 P95H/+ BM cells was significantly decreased compared to recipients repopulated with WT, p53 R248W/+, or SRSF2 P95H/+ BM cells. All recipient mice repopulated with p53 R248W/+SRSF2 P95H/+ BM cells died within 6 months and developed MDS or leukemia. Thus, we demonstrate that TP53 and SRSF2 mutations cooperate in MDS or leukemia development. In summary, we discovered that mutant p53 dysregulates pre-mRNA splicing in key regulators of inflammatory response during aging, thereby generating a chronic inflammatory microenvironment to drive MDS pathogenesis.