Biology subjects

La Greca, A.

Publications and source records attributed to La Greca, A..

13 recordsLinked to original sources

bioRxiv2026

Modulating SPARC Expression in Mesenchymal Stem Cells Improves Secretome-Mediated Skin Regeneration and Wound Repair

Mesenchymal stem cells (MSCs) have garnered significant attention over the past three decades due to their robust regenerative potential, primarily mediated by their paracrine activity by releasing soluble bioactive factors and extracellular vesicles (EVs). The MSC secretome plays a pivotal role in wound healing by influencing cellular migration, inflammation, angiogenesis, extracellular matrix (ECM) remodeling, and re-epithelialization. SPARC (Secreted Protein Acidic and Rich in Cysteine), a multifunctional ECM glycoprotein involved in tissue repair and remodeling, regulates key processes such as cell migration, proliferation, angiogenesis, and survival. Despite its known role in ECM dynamics, the impact of SPARC expression on the regenerative properties of MSCs remains underexplored. In this study, we hypothesized that SPARC overexpression in MSCs enhances their secretomes regenerative capacity. Using lentiviral systems, we generated SPARC-overexpressing (+SPARC) and SPARC-knockdown (KD-SPARC) MSCs to investigate SPARCs role in wound healing. Conditioned media (CM) derived from these MSCs were analyzed in vitro for their effects on human skin keratinocytes and fibroblasts. Our results revealed that SPARC expression significantly influences cell-specific migration and cell cycle. Furthermore, in an in vivo wound healing model, CM from +SPARC MSCs accelerated regeneration, while SPARC absence in MSCs CM delayed the healing process. These findings underscore the critical role of SPARC in modulating MSC secretome composition and enhancing its regenerative efficacy. This study highlights SPARC as a promising therapeutic target for the development of advanced regenerative therapies aimed at improving cutaneous wound healing outcomes.

cell biology↗

bioRxiv2025

Chromatin landscape of endometrial tumor cells of tamoxifen users is associated with loss of progestin regulation

Tamoxifen is an effective steroid estrogen receptor modulator (SERM) widely used in breast cancer treatment. It acts through competitive inhibition of natural ligand estrogen (E2) at the estrogen receptor (ER), affecting ER interactions with other nuclear receptors, chromatin modulators, genomic structural proteins and co-regulators. Although tamoxifen inhibits the progression of breast cancer, it increases the risk of endometrial cancer, particularly in postmenopausal women. Progesterone receptor (PR) and ER cistromes of endometrial adenocarcinoma Ishikawa cells treated with ovarian hormones showed that ER binding sites are shared by PR at open sites of chromatin. This could explain, in part, progestin regulation of estrogen effects on ER/PR positive endometrial cancer cells. Here, we compared ER cistromes from endometrial tumors of tamoxifen users and non-users with ER and PR cistromes from Ishikawal model to evaluate how SERM exposure affects ER-binding signature. Non-user ER cistrome is closer to R5020-treated Ishikawa PR cistrome, while tamoxifen users ER cistrome is similar to estradiol-treated Ishikawa ER cistrome. The subset of ERbs of non-users regulates inflammatory signaling pathway genes. In contrast, the subset of ERbs of tamoxifen users modulates early estrogen response pathway genes, such as the oncogene NRIP1. This suggests that exposure to tamoxifen redirects ER binding toward genomic regions typically responsive to estrogen, whereas ER binding in non-users retains features associated with PR-occupied regulatory sites. These results provide an example of how SERM treatment may remodel endometrial tumor epigenetic landscape, highlighting context-dependent genomic responses that should be considered to prevent deregulation of endometrial tissue under tamoxifen exposure.

cancer biology↗

bioRxiv2024

SHEN-LONG, a novel cardiac regulatory locus, regulates cardiac master transcription factor NKX2-5 in pluripotent stem cell derived cardiomyocytes

Cardiac development is a finely regulated process, transforming undifferentiated cells into the specialized cell components of the heart. Super-Enhancers (SE), clusters of enhancers densely populated with transcription factors, play a central role in cell fate decisions, including cardiogenesis. In this work, we studied a cardiac SE in chr3q25.31. This locus also contains a cardiac specific long non-coding RNA: LINC00881 (LINC881). We termed this region of interest SHEN-LONG (Super Heart ENhancer and LONG non-coding LINC881), and selected it for further functional characterization. Our analysis revealed SHEN-LONG is a hotspot of cardiac kernel transcription factor binding sites. Cardiac differentiation of human iPSC with partial Knock Out of SHEN-LONG resulted in cardiomyocytes with reduced NKX2-5 expression, a master cardiac transcription factor, suggesting a significant role of SHEN-LONG in this process. Whole transcriptome sequencing of SHEN-LONG KO cardiomyocytes produced 134 differentially expressed genes located at great distances (>4Mb) emphasizing its long-range functional impact. A regulation mediated essentially by the SE and not by LINC881 was confirmed when LINC881 overexpression did not recover NKX2-5 expression. These findings provide insights into a novel NKX2-5 regulatory mechanism. The knowledge gained in this work may pave the way for advances in therapeutic interventions for cardiovascular disorders.

molecular biology↗

bioRxiv2023

The transcription factor OCT6 promotes the dissolution of the naive pluripotent state by repressing Nanog and activating a formative state gene regulatory network.

Animal development relies on complex gene regulatory networks (GRNs) that govern the nearly irreversible changes that occur during cell differentiation. In this work we aimed to determine key transcription factors (TFs) associated with the dissolution of the naive pluripotent state and the acquisition of a formative identity. We identified OCT6 as one of the earliest TFs induced during the onset of mouse embryonic stem cell (mESCs) differentiation. To investigate its role, we generated an Oct6 knockout mESC line, which failed to acquire the characteristic cell morphology associated with the formative state. Transcriptome analysis of differentiating cells revealed nearly 300 differentially expressed genes compared to wild-type cells, including pluripotency TFs Nanog, Klf2, Nr5a2, Prdm14, and Esrrb, that failed to correctly downregulate. Notably, premature expression of OCT6 in naive cells triggered a rapid morphological transformation mirroring differentiation, accompanied by self-induction of Oct6 and expression of TFs such as Sox3, Zic2/3, Foxp1, as well as the formative genes Dnmt3A and FGF5. Strikingly, the majority of OCT6 expressing cells did not express NANOG. Gene expression and single molecule RNA-FISH analysis confirmed that this regulation was at the transcriptional level. Collectively, our results establish OCT6 as a key TF in the dissolution of the naive pluripotent state and support a model where Oct6 and Nanog form a double negative feedback loop which could act as a toggle switch important for the transition to the formative state. HighlightsO_LIOct6 is rapidly induced as mESCs exit ground state pluripotency. C_LIO_LILoss of OCT6 negatively affects the transition to formative pluripotency. C_LIO_LIPremature expression of OCT6 in mESCs is sufficient to induce a formative-like phenotype. C_LIO_LIOCT6 and NANOG repress each other forming a double negative feedback loop. C_LI

developmental biology↗

bioRxiv2022

In silico labeling enables kinetic myelination assay in brightfield

Recent advances with deep neural networks have shown the feasibility of acquiring brightfield images with transmitted light and applying in-silico labeling to predict fluorescent images. We have developed a novel in-silico labeling method based on a generative adversarial network and outperforms the state-of-the-art Unet method in generating realistic fluorescent images and quantitatively recapitulating real staining signals, as demonstrated in a complex co-culture myelination assay. Furthermore, we have performed the assay in live mode with multiple kinetic points, applied in-silico labeling to predict fluorescent images from brightfield and quantified the kinetic phenotypic changes. Thus, the proposed approach provides a potential tool to study the kinetics of cellular phenotypic changes with brightfield imaging.

cell biology↗

bioRxiv2021

Small extracellular vesicles from iPSC-MSC lose their regenerative potential upon UV-C irradiation

Mesenchymal Stem Cells derived from induced Pluripotent Stem cells (iPSC-MSC) have become a promising alternative to classical Mesenchymal Stem Cells in regenerative medicine. Their properties -as immunomodulatory and regenerative capacities-are in part due to the secretion of Extracellular Vesicles (EVs). Small EVs (sEVs) with sizes that range from 50 to 120 nm contain proteins, lipids, and nucleic acids that exert a role in cellular communication. Their content will depend on the cell of origin and its physiological state, thus the message they convey might change in response to changes in cellular conditions. In particular, the DNA damage response (DDR) has been reported to modulate sEVs secretion. In this work, we analyze how UV-C radiation upon iPSC-MSC alter sEVs secretion, cargo and bystander effect. Here, we confirm that UV-C radiation causes DDR in a dose dependent manner. In addition, we found that UV-C induced stress did not modulate the expression of genes that participate in sEVs biogenesis pathway. Consequently, we found that the amount of sEVs secreted by radiated and non-irradiated cells remained stable. However, sEVs from radiated cells were unable to promote cell migration in their target cells. Moreover, a label-free proteomic analysis revealed that UV-C induced DDR produces sEVs with an altered cargo, rich in migration-inhibiting proteins, and resulting in a less stromal-oriented repertoire.

cell biology↗

bioRxiv2021

Effects of bacterial lipopolysaccharide and Shiga Toxin on induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells

BackgroundMesenchymal Stem Cells can be activated and respond to different bacterial toxins. Lipopolysaccharides (LPS) and Shiga Toxin (Stx) are the two main bacterial toxins present in Hemolytic Uremic Syndrome (HUS) that cause endothelial damage. In this work we aimed to study the response of iPSC-MSC to LPS and/or Stx and its effect on the restoration of injured endothelial cells. MethodsiPSC-MSC were used as a source of mesenchymal stem cells (MSC) and Human Microvascular Endothelial Cells-1 (HMEC-1) as a source of endothelial cells. iPSC-MSC were treated or not with LPS and/or Stx. For some experiments, Conditioned Media (CM) were collected from each plate and incubated with an anti-Stx antibody to block the direct effect of Stx, or Polymyxin to block the direct effect of LPS. In CM from both treatments, anti-Stx and Polymyxin were used. Results are expressed as mean {+/-} S.E.M. Significant differences (p<0.05) were identified using one way analysis of variance (ANOVA) and Bonferronis Multiple comparison test. ResultsThe results obtained showed that LPS induced a pro-inflammatory profile on iPSC-MSC, but not Stx, even though they expressed Gb3 receptor. Moreover, LPS induced on iPSC-MSC an increment in migration and adhesion to gelatin substrate. Also, the addition of CM of iPSC-MSC treated with LPS+Stx, decreased the capacity of HMEC-1 to close a wound, and did not favor the formation of new tubes. Proteomic analysis of iPSC-MSC treated with LPS and/or Stx revealed specific protein secretion patterns that support many of the functional results described here. ConclusionsIn conclusion, these results suggest that iPSC-MSC activated by LPS acquired a pro-inflammatory profile that induces migration and adhesion to extracellular matrix proteins (ECM), but the combination LPS+Stx decreased the repair of endothelial damage. The importance of this work is that it provides knowledge to understand the context in which iPSC-MSC could benefit or not the restoration of tissue injury, taking into account that the inflammatory context in response to a particular bacterial toxin is relevant for iPSC-MSC immunomodulation.

immunology↗

bioRxiv2021

HLA-G gene editing: a novel therapeutic alternative in cancer immunotherapy

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial. In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G - cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.

cancer biology↗

bioRxiv2020

Single cell transfection of human induced pluripotent stem cells using a droplet-based microfluidic system.

Microfluidic tools have recently made possible many advances in biological and biomedical research. Research fields such as Physics, Engineering, Chemistry and Biology have combined to produce innovation in Microfluidics which has positively impacted on areas as diverse as nucleotide sequence, functional genomics, single-cell studies, single molecules assays, and biomedical diagnostics. Among these areas regenerative medicine and stem cells have benefited from Microfluidics due to these tools have had a profound impact on their applications. In the study, we present a high-performance droplet-based system for transfecting individual human-induced pluripotent stem cells. We show that this system has great efficiency in single cells and captured droplets, similar to other microfluidic methods and lower cost. We demonstrate that this microfluidic approach can be associated with the PiggyBac transposase-based system to increase its transfection efficiency. Our results provide a starting point for subsequent applications in more complex transfection systems, single-cell differentiation interactions, cell subpopulations, cell therapy, among other potential applications.

bioengineering↗

bioRxiv2020

Downregulation of E-cadherin in pluripotent stem cells triggers partial EMT

Epithelial to mesenchymal transition (EMT) is a critical cellular process that has been well characterized during embryonic development and cancer metastasis and it also is implicated in several physiological and pathological events including embryonic stem cell differentiation. During early stages of differentiation, human embryonic stem cells pass through EMT where deeper morphological, molecular and biochemical changes occur. Though initially considered as a decision between two states, EMT process is now regarded as a fluid transition where cells exist on a spectrum of intermediate states. In this work, using a CRISPR interference system in human embryonic stem cells, we describe a molecular characterization of the effects of downregulation of E-cadherin, one of the main initiation events of EMT, as a unique start signal. Our results suggest that the decrease and delocalization of E-cadherin causes an incomplete EMT where cells retain their undifferentiated state while expressing several characteristics of a mesenchymal-like pheno-type. Namely, we found that E-cadherin downregulation induces SNAI1 and SNAI2 upregulation, promotes MALAT1 and LINC-ROR downregulation, modulates the expression of tight junction occludin 1 and gap junction connexin 43, increases human embryonic stem cells migratory capacity and delocalize b-catenin. Altogether, we believe our results provide a useful tool to model the molecular events of an unstable intermediate state and further identify multiple layers of molecular changes that occur during partial EMT.

cell biology↗

bioRxiv2020

celldeath: a tool for simple detection of cell death in transmitted light microscopy images by visual deep learning analysis

Cell death experiments are routinely done in many labs around the world, these experiments are the backbone of many assays for drug development. Cell death detection is usually performed in many ways, and requires time and reagents. However, cell death is preceded by slight morphological changes in cell shape and texture. In this paper, we trained a neural network to classify cells undergoing cell death. We found that the network was able to highly predict cell death after one hour of exposure to camptothecin. Moreover, this prediction largely outperforms human ability. Finally, we provide a simple python tool that can broadly be used to detect cell death.

cell biology↗

bioRxiv2019

Higher-order chromatin organization defines PR and PAX2 binding to regulate endometrial cancer cell gene expression

Estrogen (E2) and Progesterone (Pg), via their specific receptors (ER and PR respectively), are major determinants in the development and progression of endometrial malignancies. Here, we have studied how E2 and the synthetic progestin R5020 affect genomic functions in Ishikawa endometrial cancer cells. Using ChIPseq in cells exposed to the corresponding hormones, we identified cell specific binding sites for ER (ERbs) and PR (PRbs), which mostly correspond to independent sites but both adjacent to sites bound by PAX2. Analysis of long-range interactions by Hi-C showed enrichment of regions co-bound by PR and PAX2 inside TADs that contain differentially progestin-regulated genes. These regions, which we call "progestin control regions" (PgCRs), exhibit an open chromatin state prior to the exposure to the hormone. Our observations suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with partner transcription factors to PgCRs, compartmentalizing hormone-independent open chromatin.

genomics↗

bioRxiv2019

Analysis of the expression of PIWI-interacting RNAs during cardiac differentiation of human pluripotent stem cells

PIWI-interacting RNAs (piRNAs) are a class of non-coding RNAs initially thought to be restricted almost exclusively to germ line cells. In recent years, accumulating evidence has demonstrated that piRNAs are actually expressed in somatic cells like pluripotent, neural, cardiac and even cancer cells. However, controversy still remains around the existence and function of somatic piRNAs. Using small RNA-seq samples from H9 pluripotent stem cells differentiated to mesoderm progenitors and cardiomyocytes we identified the expression of 447 piRNAs, of which 241 were detected in pluripotency, 218 in mesoderm and 171 in cardiac cells. The majority of them originated from the sense strand of protein coding and lncRNAs genes in all stages of differentiation, though no evidences for secondary piRNAs (ping-pong loop) were found. Genes hosting piRNAs in cardiac samples were related to critical biological processes in the heart, like contraction and cardiac muscle development. Our results indicate that somatic piRNAs might have a role in fine-tuning the expression of genes involved in the differentiation of pluripotent cells to cardiomyocytes.

molecular biology↗