State of the Art and New Trends from the Second International StemNet Meeting.

The Second International StemNet (Federation of Stem Cell Research Associations) meeting took place on 18-20 October 2023 in Brescia (Italy), with the support of the University of Brescia and the Zooprophylactic Institute of Lombardy and Emilia Romagna. The program of the meeting was articulated in nine sections: (1) Biomedical Communication in Italy: Critical Aspects; (2) StemNet Next Generation Session; (3) Cell-Free Therapies; (4) Tips and Tricks of Research Valorisation; (5) Stem Cells and Cancer; (6) Stem Cells in Veterinary Applications; (7) Stem Cells in Clinical Applications; (8) Organoids and 3D Systems; (9) induced pluripotent stem cells (iPCS) and Gene Therapy. National and International speakers presented their scientific works, inspiring debates and discussions among the attendees. The participation in the meeting was high, especially because of the young researchers who animated all the sessions and the rich poster session.

Protective effects of EVs/exosomes derived from permanently growing human MSC on primary murine ALS motor neurons.

In recent years, the neuroprotective potential of mesenchymal stroma-/stem-like cells (MSC) as well as of MSC-derived extracellular vesicles (EVs) like exosomes has been intensively explored. This included preclinical evaluation regarding treatment of neurodegenerative disorders such as the fatal motor neuron disease amyotrophic Lateral Sclerosis (ALS). Several studies have reported that MSC-derived exosomes can stimulate tissue regeneration and reduce inflammation. MSC release EVs and trophic factors and thereby modify cell-to-cell communication. These cell-free products may protect degenerating motor neurons (MNs) and represent a potential therapeutic approach for ALS. In the present study we investigated the effects of exosomes derived from a permanently growing MSC line on both, wild type and ALS (SOD1G93A transgenic) primary motor neurons. Following application in a normal and stressed environment we could demonstrate beneficial effects of MSC exosomes on neurite growth and morphology indicating the potential for further preclinical evaluation and clinical therapeutic development. Investigation of gene expression profiles detected transcripts of several antioxidant and anti-inflammatory genes in MSC exosomes. Characterization of their microRNA (miRNA) content revealed miRNAs capable of regulating antioxidant and anti-apoptotic pathways.

Why do certain cancer cells alter functionality and fuse?

Cancer cell fusion represents a rare event. However, the surviving cancer hybrid cells after a post-hybrid selection process (PHSP) can overgrow other cancer cells by exhibiting a proliferation advantage and/or expression of cancer stem-like properties. Addition of new tumor properties during hetero-fusion of cancer cells e.g. with mesenchymal stroma-/stem-like cells (MSC) contribute to enhanced tumor plasticity via acquisition of new/altered functionalities. This provides new avenues for tumor development and metastatic behavior. Consequently, the present review article will also address the question as to whether cancer cell fusion represents a general and possibly evolutionary-conserved program or rather a random process?

Intrinsic signalling factors associated with cancer cell-cell fusion.

Cellular fusion e.g. between cancer cells and normal cells represents a stepwise process that is tightly regulated. During a pre-hybrid preparation program somatic cells and/or cancer cells are promoted to a pro-fusogenic state as a prerequisite to prepare a fusion process. A pro-fusogenic state requires significant changes including restructure of the cytoskeleton, e.g., by the formation of F-actin. Moreover, distinct plasma membrane lipids such as phosphatidylserine play an important role during cell fusion. In addition, the expression of distinct fusogenic factors such as syncytins and corresponding receptors are of fundamental importance to enable cellular mergers. Subsequent hybrid formation and fusion are followed by a post-hybrid selection process. Fusion among normal cells is important and often required during organismal development. Cancer cells fusion appears more rarely and is associated with the generation of new cancer hybrid cell populations. These cancer hybrid cells contribute to an elevated tumour plasticity by altered metastatic behaviour, changes in therapeutic and apoptotic responses, and even in the formation of cancer stem/ initiating cells. While many parts within this multi-step cascade are still poorly understood, this review article predominantly focusses on the intracellular necessities for fusion among cancer cells or with other cell populations of the tumour microenvironment. Video Abstract.

Extracellular Events Involved in Cancer Cell-Cell Fusion.

Fusion among different cell populations represents a rare process that is mediated by both intrinsic and extracellular events. Cellular hybrid formation is relayed by orchestrating tightly regulated signaling pathways that can involve both normal and neoplastic cells. Certain important cell merger processes are often required during distinct organismal and tissue development, including placenta and skeletal muscle. In a neoplastic environment, however, cancer cell fusion can generate new cancer hybrid cells. Following survival during a subsequent post-hybrid selection process (PHSP), the new cancer hybrid cells express different tumorigenic properties. These can include elevated proliferative capacity, increased metastatic potential, resistance to certain therapeutic compounds, and formation of cancer stem-like cells, all of which characterize significantly enhanced tumor plasticity. However, many parts within this multi-step cascade are still poorly understood. Aside from intrinsic factors, cell fusion is particularly affected by extracellular conditions, including an inflammatory microenvironment, viruses, pH and ionic stress, hypoxia, and exosome signaling. Accordingly, the present review article will primarily highlight the influence of extracellular events that contribute to cell fusion in normal and tumorigenic tissues.

Human MSC-Derived Exosomes Reduce Cellular Senescence in Renal Epithelial Cells.

Cellular senescence of renal tubular cells is associated with chronic diseases and age-related kidney disorders. Therapies to antagonize senescence are, therefore, explored as novel approaches in nephropathy. Exosomes derived from human mesenchymal stroma-/stem-like cells (MSC) entail the transfer of multiple bioactive molecules, exhibiting profound regenerative potential in various tissues, including therapeutic effects in kidney diseases. Here, we first demonstrate that exosomes promote proliferation and reduce senescence in aged MSC cultures. For potential therapeutic perspectives in organ rejuvenation, we used MSC-derived exosomes to antagonize senescence in murine kidney primary tubular epithelial cells (PTEC). Exosome treatment efficiently reduced senescence while diminishing the transcription of senescence markers and senescence-associated secretory phenotype (SASP) factors. Concomitantly, we observed less DNA damage foci and more proliferating cells. These data provide new information regarding the therapeutic property of MSC exosomes in the development of renal senescence, suggesting a contribution to a new chapter of regenerative vehicles in senotherapy.

Cell-Cell Fusion Mediated by Viruses and HERV-Derived Fusogens in Cancer Initiation and Progression.

Cell fusion is a well-known, but still scarcely understood biological phenomenon, which might play a role in cancer initiation, progression and formation of metastases. Although the merging of two (cancer) cells appears simple, the entire process is highly complex, energy-dependent and tightly regulated. Among cell fusion-inducing and -regulating factors, so-called fusogens have been identified as a specific type of proteins that are indispensable for overcoming fusion-associated energetic barriers and final merging of plasma membranes. About 8% of the human genome is of retroviral origin and some well-known fusogens, such as syncytin-1, are expressed by human (cancer) cells. Likewise, enveloped viruses can enable and facilitate cell fusion due to evolutionarily optimized fusogens, and are also capable to induce bi- and multinucleation underlining their fusion capacity. Moreover, multinucleated giant cancer cells have been found in tumors derived from oncogenic viruses. Accordingly, a potential correlation between viruses and fusogens of human endogenous retroviral origin in cancer cell fusion will be summarized in this review.

MSC-Derived Extracellular Vesicles in Tumors and Therapy.

Exosomes derived from mesenchymal stroma-/stem-like cells (MSCs) as part of extracellular vesicles are considered cell-free biocompatible nanovesicles that promote repair activities of damaged tissues or organs by exhibiting low immunogenic and cytotoxic effects. Contributions to regenerative activities include wound healing, maintenance of stem cell niches, beneficial regenerative effects in various diseases, and reduction of senescence. However, the mode of action in MSC-derived exosomes strongly depends on the biological content like different regulatory microRNAs that are determined by the tissue origin of MSCs. In tumors, MSCs use indirect and direct pathways in a communication network to interact with cancer cells. This leads to mutual functional changes with the acquisition of an aberrant tumor-associated MSC phenotype accompanied by altered cargo in the exosomes. Consequently, MSC-derived exosomes either from normal tissue-originating MSCs or from aberrant tumor-associated MSCs can confer different actions on tumor development. These processes exhibiting tumor-inhibitory and tumor-supportive effects with a focus on exosome microRNA content will be discriminated and discussed within this review.

Hybrid Formation and Fusion of Cancer Cells In Vitro and In Vivo.

The generation of cancer hybrid cells by intra-tumoral cell fusion opens new avenues for tumor plasticity to develop cancer stem cells with altered properties, to escape from immune surveillance, to change metastatic behavior, and to broaden drug responsiveness/resistance. Genomic instability and chromosomal rearrangements in bi- or multinucleated aneuploid cancer hybrid cells contribute to these new functions. However, the significance of cell fusion in tumorigenesis is controversial with respect to the low frequency of cancer cell fusion events and a clonal advantage of surviving cancer hybrid cells following a post-hybrid selection process. This review highlights alternative processes of cancer hybrid cell development such as entosis, emperipolesis, cannibalism, therapy-induced polyploidization/endoreduplication, horizontal or lateral gene transfer, and focusses on the predominant mechanisms of cell fusion. Based upon new properties of cancer hybrid cells the arising clinical consequences of the subsequent tumor heterogeneity after cancer cell fusion represent a major therapeutic challenge.

Cancer Cell Fusion and Post-Hybrid Selection Process (PHSP).

Fusion of cancer cells either with other cancer cells (homotypic fusion) in local vicinity of the tumor tissue or with other cell types (e.g., macrophages, cancer-associated fibroblasts (CAFs), mesenchymal stromal-/stem-like cells (MSC)) (heterotypic fusion) represents a rare event. Accordingly, the clinical relevance of cancer-cell fusion events appears questionable. However, enhanced tumor growth and/or development of certain metastases can originate from cancer-cell fusion. Formation of hybrid cells after cancer-cell fusion requires a post-hybrid selection process (PHSP) to cope with genomic instability of the parental nuclei and reorganize survival and metabolic functionality. The present review dissects mechanisms that contribute to a PHSP and resulting functional alterations of the cancer hybrids. Based upon new properties of cancer hybrid cells, the arising clinical consequences of the subsequent tumor heterogeneity after cancer-cell fusion represent a major therapeutic challenge. However, cellular partners during cancer-cell fusion such as MSC within the tumor microenvironment or MSC-derived exosomes may provide a suitable vehicle to specifically address and deliver anti-tumor cargo to cancer cells.

Spontaneous Fusion of MSC with Breast Cancer Cells Can Generate Tumor Dormancy.

Direct cellular interactions of MDA-MB-231cherry breast cancer cells with GFP-transduced human mesenchymal stroma/stem-like cells (MSCGFP) in a co-culture model resulted in spontaneous cell fusion by the generation of MDA-MSC-hyb5cherry GFP breast cancer hybrid cells. The proliferative capacity of MDA-MSC-hyb5 cells was enhanced about 1.8-fold when compared to the parental MDA-MB-231cherry breast cancer cells. In contrast to a spontaneous MDA-MB-231cherry induced tumor development in vivo within 18.8 days, the MDA-MSC-hyb5 cells initially remained quiescent in a dormancy-like state. At distinct time points after injection, NODscid mice started to develop MDA-MSC-hyb5 cell-induced tumors up to about a half year later. Following tumor initiation, however, tumor growth and formation of metastases in various different organs occurred rapidly within about 10.5 days. Changes in gene expression levels were evaluated by RNA-microarray analysis and revealed certain increase in dormancy-associated transcripts in MDA-MSC-hyb5. Chemotherapeutic responsiveness of MDA-MSC-hyb5 cells was partially enhanced when compared to MDA-MB-231 cells. However, some resistance, e.g., for taxol was detectable in cancer hybrid cells. Moreover, drug response partially changed during the tumor development of MDA-MSC-hyb5 cells; this suggests the presence of unstable in vivo phenotypes of MDA-hyb5 cells with increased tumor heterogeneity.

Effect of SSRI exposure on the proliferation rate and glucose uptake in breast and ovary cancer cell lines.

Breast cancer is the most prevalent malignancy amongst women worldwide while ovarian cancer represents the leading cause of death among gynecological malignancies. Women suffering from these cancers displayed heightened rates of major depressive disorder, and antidepressant treatment with selective serotonin reuptake inhibitors (SSRIs) is frequently recommended. Recently, narrative reviews and meta-analyses showed increased recurrence risks and mortality rates in SSRI-treated women with breast and ovarian cancer. We therefore examined whether three commonly prescribed SSRIs, fluoxetine, sertraline and citalopram, affect proliferation or glucose uptake of human breast and ovarian cancer cell lines characterized by different malignancies and metastatic potential. SSRI treatment or serotonin stimulation with therapeutically relevant concentrations over various time periods revealed no consistent dose- or time-dependent effect on proliferation rates. A marginal, but significant increase in glucose uptake was observed in SK-OV-3 ovarian cancer cells upon fluoxetine or sertraline, but not citalopram treatment. In three breast cancer cell lines and in two additional ovarian cancer cell lines no significant effect of SSRIs on glucose uptake was observed. Our data suggest that the observed increase in recurrence- and mortality rates in SSRI-treated cancer patients is unlikely to be linked to antidepressant therapies.

Impact of the Tumor Microenvironment on Tumor Heterogeneity and Consequences for Cancer Cell Plasticity and Stemness.

Tumor heterogeneity is considered the major cause of treatment failure in current cancer therapies. This feature of solid tumors is not only the result of clonal outgrowth of cells with genetic mutations, but also of epigenetic alterations induced by physical and chemical signals from the tumor microenvironment (TME). Besides fibroblasts, endothelial and immune cells, mesenchymal stroma/stem-like cells (MSCs) and tumor-associated macrophages (TAMs) intimately crosstalk with cancer cells and can exhibit both anti- and pro-tumorigenic effects. MSCs can alter cancer cellular phenotypes to increase cancer cell plasticity, eventually resulting in the generation of cancer stem cells (CSCs). The shift between different phenotypic states (phenotype switching) of CSCs is controlled via both genetic programs, such as epithelial-mesenchymal transdifferentiation or retrodifferentiation, and epigenetic alterations triggered by signals from the TME, like hypoxia, spatial heterogeneity or stromal cell-derived chemokines. Finally, we highlight the role of spontaneous cancer cell fusion with various types of stromal cells. i.e., MSCs in shaping CSC plasticity. A better understanding of cell plasticity and phenotype shifting in CSCs is a prerequisite for exploiting this phenomenon to reduce tumor heterogeneity, thereby improving the chance for therapy success.

The Intimate Relationship Among EMT, MET and TME: A T(ransdifferentiation) E(nhancing) M(ix) to Be Exploited for Therapeutic Purposes.

Intratumoral heterogeneity is considered the major cause of drug unresponsiveness in cancer and accumulating evidence implicates non-mutational resistance mechanisms rather than genetic mutations in its development. These non-mutational processes are largely driven by phenotypic plasticity, which is defined as the ability of a cell to reprogram and change its identity (phenotype switching). Tumor cell plasticity is characterized by the reactivation of developmental programs that are closely correlated with the acquisition of cancer stem cell properties and an enhanced potential for retrodifferentiation or transdifferentiation. A well-studied mechanism of phenotypic plasticity is the epithelial-mesenchymal transition (EMT). Current evidence suggests a complex interplay between EMT, genetic and epigenetic alterations, and clues from the tumor microenvironment in cell reprogramming. A deeper understanding of the connections between stem cell, epithelial-mesenchymal, and tumor-associated reprogramming events is crucial to develop novel therapies that mitigate cell plasticity and minimize the evolution of tumor heterogeneity, and hence drug resistance. Alternatively, vulnerabilities exposed by tumor cells when residing in a plastic or stem-like state may be exploited therapeutically, i.e., by converting them into less aggressive or even postmitotic cells. Tumor cell plasticity thus presents a new paradigm for understanding a cancer's resistance to therapy and deciphering its underlying mechanisms.

Altered Tumor Plasticity after Different Cancer Cell Fusions with MSC.

While cell fusion demonstrates an important pathway during tissue development and regeneration of distinct organs, this process can also contribute to pathophysiological phenotypes during tumor progression. Hybrid cell formation after heterofusion between cancer cells and various other cell types within the tumor microenvironment is observed in vitro and in vivo. In particular, mesenchymal stroma/stem-like cells (MSC) perform diverse levels of communication with cancer cells by exhibiting anti- and pro-tumorigenic effects. During these cellular interactions, MSC can eventually fuse with cancer cells. Thereby, the newly generated disparate hybrid populations display aneuploidy associated with chromosomal instability. Based upon a subsequent post-hybrid selection process (PHSP), fused cancer cells can undergo apoptosis/necroptosis, senescence, dormancy, or a proliferative state by acquisition of new properties. Consequently, PHSP-surviving hybrid cancer cells demonstrate altered functionalities within the tumor tissue. This is accompanied by changes in therapeutic responsiveness and a different metastatic behavior. Accordingly, enhanced tumor plasticity interferes with successful therapeutic interventions and aggravates patient prognoses. The present review article focusses on fusion of MSC with different human cancer cells, in particular breast cancer populations and resulting characteristics of various cancer hybrid cells. Moreover, some mechanisms of cancer cell fusion are discussed together with multiple PHSP pathways.

Anti-Tumor Effects of Exosomes Derived from Drug-Incubated Permanently Growing Human MSC.

Similar to growth-limited human primary cultures of mesenchymal stroma/stem-like cells (MSC), the continuously proliferating human MSC544 cell line produced extracellular vesicles as characterized by expression of the tetraspanin molecules CD9, CD63, and CD81. Release of these particles was predominantly detectable during continuous cell growth of MSC544 in contrast to confluency-mediated transient growth arrest. For therapeutic use, these particles were isolated from proliferating MSC544 after taxol treatment and applied to different cancer cell cultures. A pronounced cytotoxicity of lung, ovarian, and breast cancer cells was observed primarily with taxol-loaded exosomes, similar to the effects displayed by application of taxol substance. While these findings suggested pronounced cancer cell targeting of MSC544 exosomes, a tumor therapeutic approach was performed using a mouse in vivo breast cancer model. Thus, intravenous injection of taxol-loaded MSC544 exosomes displayed superior tumor-reducing capabilities as compared to application of taxol exosomes by oral gavage. To broaden this therapeutic spectrum, epirubicin was applied to MSC544, and the derived exosomes likewise exhibited significant cytotoxic effects in different cancer cell cultures. These findings suggest an unlimited source for large-scale exosome production with reproducible quality to enable variable drug targeting of tumors or other diseases.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

Role of MSC in the Tumor Microenvironment.

The tumor microenvironment represents a dynamically composed matrix in which tissue-associated cancer cells are embedded together with a variety of further cell types to form a more or less separate organ-like structure. Constantly mutual interactions between cells of the tumor microenvironment promote continuous restructuring and growth in the tumor. A distinct organization of the tumor stroma also facilitates the formation of transient cancer stem cell niches, thereby contributing to progressive and dynamic tumor development. An important but heterogeneous mixture of cells that communicates among the cancer cells and the different tumor-associated cell types is represented by mesenchymal stroma-/stem-like cells (MSC). Following recruitment to tumor sites, MSC can change their functionalities, adapt to the tumor's metabolism, undergo differentiation and synergize with cancer cells. Vice versa, cancer cells can alter therapeutic sensitivities and change metastatic behavior depending on the type and intensity of this MSC crosstalk. Thus, close cellular interactions between MSC and cancer cells can eventually promote cell fusion by forming new cancer hybrid cells. Consequently, newly acquired cancer cell functions or new hybrid cancer populations enlarge the plasticity of the tumor and counteract successful interventional strategies. The present review article highlights some important features of MSC within the tumor stroma.

Reversible Growth-Arrest of a Spontaneously-Derived Human MSC-Like Cell Line.

Life cycle limitation hampers the production of high amounts of primary human mesenchymal stroma-/stem-like cells (MSC) and limits cell source reproducibility for clinical applications. The characterization of permanently growing MSC544 revealed some differentiation capacity and the simultaneous presence of known MSC markers CD73, CD90, and CD105 even after continuous long-term culture for more than one year and 32 passages. The expression of CD13, CD29, CD44, and CD166 were identified as further surface proteins, all of which were also simultaneously detectable in various other types of primary MSC populations derived from the umbilical cord, bone marrow, and placenta suggesting MSC-like properties in the cell line. Proliferating steady state MSC544 exhibited immune-modulatory activity similar to a subpopulation of long-term growth-inhibited MSC544 after 189d of continuous culture in confluency. This confluent connective cell layer with fibroblast-like morphology can spontaneously contract and the generated space is subsequently occupied by new cells with regained proliferative capacity. Accordingly, the confluent and senescence-associated beta-galactosidase-positive MSC544 culture with about 95% G0/G1 growth-arrest resumed re-entry into the proliferative cell cycle within 3d after sub-confluent culture. The MSC544 cells remained viable during confluency and throughout this transition which was accompanied by marked changes in the release of proteins. Thus, expression of proliferation-associated genes was down-modulated in confluent MSC544 and re-expressed following sub-confluent conditions whilst telomerase (hTERT) transcripts remained detectable at similar levels in both, confluent growth-arrested and proliferating MSC544. Together with the capability of connective cell layer formation for potential therapeutic approaches, MSC544 provide a long term reproducible human cell source with constant properties.

Potential Role of MSC/Cancer Cell Fusion and EMT for Breast Cancer Stem Cell Formation.

Solid tumors comprise of maturated cancer cells and self-renewing cancer stem-like cells (CSCs), which are associated with various other nontumorigenic cell populations in the tumor microenvironment. In addition to immune cells, endothelial cells, fibroblasts, and further cell types, mesenchymal stroma/stem-like cells (MSC) represent an important cell population recruited to tumor sites and predominantly interacting with the different cancer cells. Breast cancer models were among the first to reveal distinct properties of CSCs, however, the cellular process(es) through which these cells are generated, maintained, and expanded within neoplastic tissues remains incompletely understood. Here, we discuss several possible scenarios that are not mutually exclusive but may even act synergistically: fusion of cancer cells with MSC to yield hybrid cells and/or the induction of epithelial-mesenchymal transition (EMT) in breast cancer cells by MSC, which can relay signals for retrodifferentiation and eventually, the generation of breast CSCs (BCSCs). In either case, the consequences may be promotion of self-renewal capacity, tumor cell plasticity and heterogeneity, an increase in the cancer cells' invasive and metastatic potential, and the acquisition of resistance mechanisms towards chemo- or radiotherapy. While specific signaling mechanisms involved in each of these properties remain to be elucidated, the present review article focusses on a potential involvement of cancer cell fusion and EMT in the development of breast cancer stem cells.