Mesenchymal stem cells derived from adipose tissue and umbilical cord reveal comparable efficacy upon radiation-induced colorectal fibrosis in rats.

Chemoradiotherapy (CRT) and radiotherapy (RT) have served as anticancer treatments and neoadjuvant therapies for conquering multimodal rectal cancers including colorectal carcinoma (CRC), yet the concomitant radiation-induced colorectal fibrosis (RICF) has caused chronic toxicity and stenosis in the colorectal mucosa of patients. Mesenchymal stem/stromal cells (MSCs) with unique bidirectional immunoregulation and anti-fibrotic effect have been recognized as splendid sources for regenerative purposes including intestinal diseases. Herein, we are aiming to verify the feasibility and variations of MSC-based cytotherapy for the remission of RICF from the pathological features and the potential impact upon the transcriptomic signatures of RICF rats. For the purpose, we utilized our well-established RICF Sprague-Dawley (SD) rats by radiation for five weeks, and conducted consecutive intraperitoneal injection of two distinct MSCs for treatment, including MSCs derived from adult adipose tissue (AD-MSCs) and perinatal umbilical cord (UC-MSCs). On the one hand, the efficacy of AD-MSCs and UC-MSCs was assessed by diverse indicators, including weight change, pathological detections (e.g., H&E staining, Masson staining, EVG staining, IF staining, and IHC staining), and proinflammatory and fibrotic factor expression. On the other hand, we turned to RNA-sequencing (RNA-SEQ) and multifaceted bioinformatics analyses (e.g., GOBP, Venn Map, KEGG, and GSEA) to compare the impact of AD-MSC and UC-MSC treatment upon the gene expression profiling and genetic variations. RICF rats after consecutive AD-MSC and UC-MSC administration revealed comparable remission in histopathogenic features and significant suppression of diverse proinflammatory and fibrotic factors expression. Meanwhile, RICF rats after both MSC treatment revealed decrease and variations in the alterations in diverse gene expression and somatic mutations compared to RICF rats. Collectively, our data indicated the comparable therapeutic effect of AD-MSCs and UC-MSCs upon RICF in SD rats, together with the conservations in gene expression profiling and the diverse variations in genetic mutations. Our findings indicated the multifaceted impact of MSC infusion for the supervision of RICF both at the therapeutic and transcriptomic levels, which would provide novel references for the further evaluation and development of MSC-based regimens in future.

Intra-Articular Application of Autologous, Fat-Derived Orthobiologics in the Treatment of Knee Osteoarthritis: A Systematic Review.

The aim of this study was to review the current literature regarding the effects of intra-articularly applied, fat-derived orthobiologics (FDO) in the treatment of primary knee osteoarthritis over a mid-term follow-up period. A systematic literature search was conducted on the online databases of Scopus, PubMed, Ovid MEDLINE, and Cochrane Library. Studies investigating intra-articularly applied FDO with a minimum number of 10 knee osteoarthritis patients, a follow-up period of at least 2 years, and at least 1 reported functional parameter (pain level or Patient-Reported Outcome Measures) were included. Exclusion criteria encompassed focal chondral defects and techniques including additional arthroscopic bone marrow stimulation. In 28 of 29 studies, FDO showed a subjective improvement in symptoms (pain and Patient-Reported Outcome Measures) up to a maximum follow-up of 7.2 years. Radiographic cartilage regeneration up to 3 years postoperatively, as well as macroscopic cartilage regeneration investigated via second-look arthroscopy, may corroborate the favorable clinical findings in patients with knee osteoarthritis. The methodological heterogeneity in FDO treatments leads to variations in cell composition and represents a limitation in the current state of knowledge. However, this systematic review suggests that FDO injection leads to beneficial mid-term results including symptom reduction and preservation of the affected joint in knee osteoarthritis patients.

Amphiregulin secreted by umbilical cord Multipotent Stromal Cells protects against ferroptosis of macrophages via the Activating Transcription Factor 3-CD36 axis to alleviate endometrial fibrosis.

Endometrium fibrosis is the leading cause of uterine infertility. Macrophages participated in the occurrence and development of endometrial fibrosis. We previously reported that human umbilical cord multipotent stromal cells (hUC-MSCs) exerted their therapeutic effect in a macrophage-dependent manner in endometrial fibrosis. However precise mechanisms by which hUC-MSCs may influence macrophages in endometrial fibrosis remain largely unexplored. Here, we demonstrated that abnormal iron and lipid metabolism occurred in intrauterine adhesions (IUA) patients and murine models. Ferroptosis has been proven to contribute to the progression of fibrotic diseases. Our results revealed that pharmacological activation of ferroptosis by Erastin aggravated endometrial fibrosis, while inhibition of ferroptosis by Ferrostatin-1 ameliorated endometrial fibrosis in vivo. Moreover, ferroptosis of macrophages was significantly upregulated in endometria of IUA murine models. Of note, transcriptome profiles revealed that CD36 gene expression was significantly increased in IUA patients and immunofluorescence analysis showed CD36 protein was mainly located in macrophages. Silencing CD36 in macrophages could reverse cell ferroptosis. Dual luciferase reporter assay revealed that CD36 was the direct target of activation transcription factor 3 (ATF3). Furthermore, through establishing coculture system and IUA murine models, we found that hUC-MSCs had a protective role against macrophage ferroptosis and alleviated endometrial fibrosis related to decreased CD36 and ATF3. The effect of hUC-MSCs on macrophage ferroptosis was attributed to the upregulation of amphiregulin (AREG). Our data highlighted that macrophage ferroptosis occurred in endometrial fibrosis via the ATF3-CD36 pathway and hUC-MSCs protected against macrophage ferroptosis to alleviate endometrial fibrosis via secreting AREG. These findings provided a potential target for therapeutic implications of endometrial fibrosis.

Buccal Mesenchymal Stromal Cells as a Source of Osseointegration of Titanium Implants.

We studied the interaction of human buccal mesenchymal stem cells (MSCs) and osteoblasts differentiated from them with the surface of titanium samples. MSCs were isolated by enzymatic method from buccal fat pads. The obtained cell culture was presented by MSCs, which was confirmed by flow cytometry and differentiation into adipocytes and osteoblasts. Culturing of buccal MSCs on titanium samples was accompanied by an increase in the number of cells for 15 days and the formation of a developed network of F-actin fibers in the cells. The viability of buccal MSCs decreased by 8 days, but was restored by 15 days. Culturing of osteoblasts obtained as a result of buccal MSC differentiation on the surface of titanium samples was accompanied by a decrease in their viability and proliferation. Thus, MSCs from buccal fat pads can be used to coat implants to improve osseointegration during bone reconstruction in craniofacial surgery and dentistry. To improve the integration of osteoblasts, modification of the surface of titanium samples is required.

Comprehensive analysis of human monocyte subsets using full-spectrum flow cytometry and hierarchical marker clustering.

Introduction: Exploring monocytes' roles within the tumor microenvironment is crucial for crafting targeted cancer treatments. Methods: This study unveils a novel methodology utilizing four 20-color flow cytometry panels for comprehensive peripheral immune system phenotyping, specifically targeting classical, intermediate, and non-classical monocyte subsets. Results: By applying advanced dimensionality reduction techniques like t-distributed stochastic neighbor embedding (tSNE) and FlowSom analysis, we performed an extensive profiling of monocytes, assessing 50 unique cell surface markers related to a wide range of immunological functions, including activation, differentiation, and immune checkpoint regulation. Discussion: This in-depth approach significantly refines the identification of monocyte subsets, directly supporting the development of personalized immunotherapies and enhancing diagnostic precision. Our pioneering panel for monocyte phenotyping marks a substantial leap in understanding monocyte biology, with profound implications for the accuracy of disease diagnostics and the success of checkpoint-inhibitor therapies. Key findings include revealing distinct marker expression patterns linked to tumor progression and providing new avenues for targeted therapeutic interventions.

Adipose-derived regenerative therapies for the treatment of knee osteoarthritis.

Knee osteoarthritis is a degenerative condition with a significant disease burden and no disease-modifying therapy. Definitive treatment ultimately requires joint replacement. Therapies capable of regenerating cartilage could significantly reduce financial and clinical costs. The regenerative potential of mesenchymal stromal cells (MSCs) has been extensively studied in the context of knee osteoarthritis. This has yielded promising results in human studies, and is likely a product of immunomodulatory and chondroprotective biomolecules produced by MSCs in response to inflammation. Adipose-derived MSCs (ASCs) are becoming increasingly popular owing to their relative ease of isolation and high proliferative capacity. Stromal vascular fraction (SVF) and micro-fragmented adipose tissue (MFAT) are produced by the enzymatic and mechanical disruption of adipose tissue, respectively. This avoids expansion of isolated ASCs ex vivo and their composition of heterogeneous cell populations, including immune cells, may potentiate the reparative function of ASCs. In this editorial, we comment on a multicenter randomized trial regarding the efficacy of MFAT in treating knee osteoarthritis. We discuss the study's findings in the context of emerging evidence regarding adipose-derived regenerative therapies. An underlying mechanism of action of ASCs is proposed while drawing important distinctions between the properties of isolated ASCs, SVF, and MFAT.

Single-cell sequencing advances in research on mesenchymal stem/stromal cells.

Mesenchymal stem/stromal cells (MSCs), originating from the mesoderm, represent a multifunctional stem cell population capable of differentiating into diverse cell types and exhibiting a wide range of biological functions. Despite more than half a century of research, MSCs continue to be among the most extensively studied cell types in clinical research projects globally. However, their significant heterogeneity and phenotypic instability have significantly hindered their exploration and application. Single-cell sequencing technology emerges as a powerful tool to address these challenges, offering precise dissection of complex cellular samples. It uncovers the genetic structure and gene expression status of individual contained cells on a massive scale and reveals the heterogeneity among these cells. It links the molecular characteristics of MSCs with their clinical applications, contributing to the advancement of regenerative medicine. With the development and cost reduction of single-cell analysis techniques, sequencing technology is now widely applied in fundamental research and clinical trials. This study aimed to review the application of single-cell sequencing in MSC research and assess its prospects.

HAX1-Overexpression Augments Cardioprotective Efficacy of Stem Cell-Based Therapy Through Mediating Hippo-Yap Signaling.

Although stem/progenitor cell therapy shows potential for myocardial infarction repair, enhancing the therapeutic efficacy could be achieved through additional genetic modifications. HCLS1-associated protein X-1 (HAX1) has been identified as a versatile modulator responsible for cardio-protective signaling, while its role in regulating stem cell survival and functionality remains unknown. In this study, we investigated whether HAX1 can augment the protective potential of Sca1+ cardiac stromal cells (CSCs) for myocardial injury. The overexpression of HAX1 significantly increased cell proliferation and conferred enhanced resistance to hypoxia-induced cell death in CSCs. Mechanistically, HAX1 can interact with Mst1 (a prominent conductor of Hippo signal transduction) and inhibit its kinase activity for protein phosphorylation. This inhibition led to enhanced nuclear translocation of Yes-associated protein (YAP) and activation of downstream therapeutic-related genes. Notably, HAX1 overexpression significantly increased the pro-angiogenic potential of CSCs, as demonstrated by elevated expression of vascular endothelial growth factors. Importantly, implantation of HAX1-overexpressing CSCs promoted neovascularization, protected against functional deterioration, and ameliorated cardiac fibrosis in ischemic mouse hearts. In conclusion, HAX1 emerges as a valuable and efficient inducer for enhancing the effectiveness of cardiac stem or progenitor cell therapeutics.

Chronic Idiopathic Ulcers Mimicking Cecal Carcinoma: A Case Report.

Chronic idiopathic ulcers of the colon pose diagnostic challenges due to their elusive etiology and potential resemblance to other intestinal pathologies, such as cecal carcinoma. This case report outlines the clinical course of a 68-year-old female patient who presented to the emergency department (ED) with persistent right lower quadrant pain. Despite multiple hospital visits yielding varied diagnoses, a definitive diagnosis was only made following a laparoscopic partial colectomy, which revealed chronic idiopathic ulcers with transmural scarring and adhesions to adjacent small intestine loops. Histological examination demonstrated a substantial ulcer bed populated by inflammatory cells, including large stellate and spindled stromal cells within the granulation tissue, alongside lymphoid hyperplasia and scar tissue extending into the muscularis propria. The initial presentation of this case could easily be mistaken for appendicitis, diverticulitis, carcinoma, or irritable bowel syndrome, highlighting the significance of considering chronic idiopathic ulcers in the differential diagnosis of patients presenting with cecal masses.

IGF2BP2 regulates the proliferation and migration of endometrial stromal cells through the PI3K/AKT/mTOR signaling pathway in Hu sheep.

Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), a significant member of the conserved RNA-binding protein family, plays various roles in numerous physiological and pathological processes. However, the specific function of IGF2BP2 in regulating endometrial function in sheep remains largely unknown. In this study, we observed a significant upregulation in IGF2BP2 mRNA abundance in the endometrium during the luteal phase (LP) compared to the follicular phase (FP) in Hu sheep. The knockdown of IGF2BP2 resulted in accelerated cell proliferation and migration of Hu sheep endometrial stromal cells (ESCs). Moreover, RNA sequencing analysis revealed that genes with significantly altered expression in IGF2BP2 knockdown cells were predominantly enriched in endometrial receptivity-related signaling pathways, such as cytokine-cytokine receptor interaction, NOD-like receptor, PI3K-AKT, and JAK-STAT signaling pathway. Additionally, the knockdown of IGF2BP2 significantly increased the expression of matrix metalloprotein 9 (MMP9), vascular endothelial growth factor (VEGF), and prolactin (PRL) in ESCs. The knockdown of IGF2BP2 was also observed to stimulate the PI3K/AKT/mTOR pathway by upregulating integrin ß4 (ITGB4) expression. Notably, the downregulation of ITGB4 attenuates IGF2BP2 knockdown-induced facilitation of proliferation and migration of Hu sheep ESCs by inhibiting the PI3K/AKT/mTOR pathway. Collectively, these findings highlight the important role of IGF2BP2 in regulating endometrial function, particularly through the modulation of ESC proliferation and migration via the PI3K/AKT/mTOR pathway.

Treatment of post-allogeneic hematopoietic stem cell transplant cytopenias with sequential doses of multipotent mesenchymal stromal/stem cells.

BACKGROUND AIMS: Cytopenias after allogeneic stem cell transplantation (allo-SCT) are a common complication, the underlying pathogenic mechanisms of which remain incompletely understood. Multipotent mesenchymal stromal/stem cell (MSC) therapy has been successfully employed in the treatment of immune-related disorders and can aid in the restoration of the hematopoietic niche. METHODS: A phase II clinical trial to assess the efficacy and safety of administering four sequential doses of ex-vivo expanded bone marrow MSCs from a third-party donor to patients with persistent severe cytopenias after allo-SCT was performed. RESULTS: The overall response rate on day 90 was 75% among the 27 evaluable patients (comprising 12 complete responses, 8 partial responses, and 7 with no response). The median time to respond was 14.5 days. Responses were observed across different profiles, including single or multiple affected lineages, primary or secondary timing, and potential immune-mediated or post-infectious pathophysiology versus idiopathic origin. With a median follow-up for surviving patients of 85 months after MSC infusion, 53% of patients are alive. Notably, no adverse events related to MSC therapy were reported. CONCLUSIONS: In summary, the sequential infusion of third-party MSCs emerges as a viable and safe therapeutic option, exhibiting potential benefits for patients experiencing cytopenias following allo-SCT.

Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture.

BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.

Adipose-tissue Treg cells restrain differentiation of stromal adipocyte precursors to promote insulin sensitivity and metabolic homeostasis.

Regulatory T (Treg) cells in epidydimal visceral adipose tissue (eVAT) of lean mice and humans regulate metabolic homeostasis. We found that constitutive or punctual depletion of eVAT-Treg cells reined in the differentiation of stromal adipocyte precursors. Co-culture of these precursors with conditional medium from eVAT-Treg cells limited their differentiation in vitro, suggesting a direct effect. Transcriptional comparison of adipocyte precursors, matured in the presence or absence of the eVAT-Treg-conditioned medium, identified the oncostatin-M (OSM) signaling pathway as a key distinction. Addition of OSM to in vitro cultures blocked the differentiation of adipocyte precursors, while co-addition of anti-OSM antibodies reversed the ability of the eVAT-Treg-conditioned medium to inhibit in vitro adipogenesis. Genetic depletion of OSM (specifically in Treg) cells or of the OSM receptor (specifically on stromal cells) strongly impaired insulin sensitivity and related metabolic indices. Thus, Treg-cell-mediated control of local progenitor cells maintains adipose tissue and metabolic homeostasis, a regulatory axis seemingly conserved in humans.

Skeletal stem and progenitor cells in bone development and repair.

Bone development, growth, and repair are complex processes involving various cell types and interactions, with central roles played by skeletal stem and progenitor cells. Recent research brought new insights into the skeletal precursor populations that mediate intramembranous and endochondral bone development. Later in life, many of the cellular and molecular mechanisms determining development are reactivated upon fracture, with powerful trauma-induced signaling cues triggering a variety of postnatal skeletal stem/progenitor cells (SSPCs) residing near the bone defect. Interestingly, in this injury context, the current evidence suggests that the fates of both SSPCs and differentiated skeletal cells can be considerably flexible and dynamic, and that multiple cell sources can be activated to operate as functional progenitors generating chondrocytes and/or osteoblasts. The combined implementation of in vivo lineage tracing, cell surface marker-based selection, single-cell molecular analyses, and high-resolution in situ imaging has strongly improved our insights into the diversity and roles of developmental and reparative stem/progenitor subsets, while also unveiling the complexity of their dynamics, hierarchies, and relationships. Albeit incompletely understood at present, findings supporting lineage flexibility and possibly plasticity among sources of osteogenic cells challenge the classical dogma of a single primitive, self-renewing, multipotent stem cell driving bone tissue formation and regeneration from the apex of a hierarchical and strictly unidirectional differentiation tree. We here review the state of the field and the newest discoveries in the origin, identity, and fates of skeletal progenitor cells during bone development and growth, discuss the contributions of adult SSPC populations to fracture repair, and reflect on the dynamism and relationships among skeletal precursors and differentiated cell lineages. Further research directed at unraveling the heterogeneity and capacities of SSPCs, as well as the regulatory cues determining their fate and functioning, will offer vital new options for clinical translation toward compromised fracture healing and bone regenerative medicine.

Skeletal progenitor cells are crucial for bone development and growth, as they provide the cellular building blocks (chondrocytes and osteoblasts) that form the cartilage and bone tissues that the skeleton is composed of. In adult life, the occurrence of a bone fracture reactivates similar tissue-forming mechanisms, starting with the trauma triggering various postnatal skeletal stem/progenitor cells (SSPCs) residing near the bone defect to divide and migrate. These cells subsequently generate functional fracture-repairing cells by differentiating into mature chondrocytes and/or osteoblasts. In recent years, the combined use of various advanced research approaches and new techniques has strongly improved our insights into the origin, identity, fates, and roles of developmental and reparative skeletal stem cells and progenitor subsets. Concomitantly, this research also unveiled considerable complexity in their dynamics, diversity, hierarchies, and relationships, which is incompletely understood at present. In this review, we discuss the state of the field and the newest discoveries in the identity and roles of skeletal stem and progenitor cells mediating bone development, growth, and repair. Further research on these cell populations, including determining their exact nature, fate, and functioning, and how they can be harvested and regulated, is critical to develop new treatments for non-healing fractures.

Comparative analysis of uninduced and neuronally-induced human dental pulp stromal cells in a 6-OHDA model of Parkinson's disease.

BACKGROUND: In recent years, dental pulp stromal cells (DPSCs) have emerged as a promising therapeutic approach for Parkinson's disease (PD), owing to their inherent neurogenic potential and the lack of neuroprotective treatments for this condition. However, uncertainties persist regarding the efficacy of these cells in an undifferentiated state versus a neuronally-induced state. This study aims to delineate the distinct therapeutic potential of uninduced and neuronally-induced DPSCs in a rodent model of PD induced by 6-Hydroxydopamine (6-OHDA). METHODS: DPSCs were isolated from human teeth, characterized as mesenchymal stromal cells, and induced to neuronal differentiation. Neuronal markers were assessed before and after induction. DPSCs were transplanted into the substantia nigra pars compacta (SNpc) of rats 7 days following the 6-OHDA lesion. In vivo tracking of the cells, evaluation of locomotor behavior, dopaminergic neuron survival, and the expression of essential proteins within the dopaminergic system were conducted 7 days postgrafting. RESULTS: Isolated DPSCs exhibited typical characteristics of mesenchymal stromal cells and maintained a normal karyotype. DPSCs consistently expressed neuronal markers, exhibiting elevated expression of ßIII-tubulin following neuronal induction. Results from the animal model showed that both DPSC types promoted substantial recovery in dopaminergic neurons, correlating with enhanced locomotion. Additionally, neuronally-induced DPSCs prevented GFAP elevation, while altering DARPP-32 phosphorylation states. Conversely, uninduced DPSCs reduced JUN levels. Both DPSC types mitigated the elevation of glycosylated DAT. CONCLUSIONS: Our results suggested that uninduced DPSCs and neuronally-induced DPSCs exhibit potential in reducing dopaminergic neuron loss and improving locomotor behavior, but their underlying mechanisms differ.

The Effect of Cell Culture Passage on the Efficacy of Mesenchymal Stromal Cells as a Cell Therapy Treatment.

Background/Objective: Mesenchymal Stromal Cells (MSCs) have been considered a promising treatment for several diseases, such as cardiac injuries. Many studies have analyzed their functional properties; however, few studies have characterized MSCs through successive culture passages. The main objective of this work was to analyze the phenotype and functionality of MSCs isolated from two different sources in five culture passages to determine if the culture passage might influence the efficacy of MSCs as a cell therapy treatment. Methods: Bone Marrow (BM)-MSCs were harvested from the femur of Wistar rats (n = 17) and Adipose Tissue(AT)-MSCs were isolated from inguinal fat (n = 17). MSCs were cultured for five culture passages, and the immunophenotype was analyzed by flow cytometry, the functionality was characterized by adipogenic, osteogenic, and chondrogenic differentiation assays, and cytokine secretion capacity was determined through the quantification of the Vascular Endothelial Growth-Factor, Fibroblast Growth-Factor2, and Transforming Growth-Factorß1 in the cell supernatant. The ultrastructure of MSCs was analyzed by transmission electron microscopy. Results: BM-MSCs exhibited typical phenotypes in culture passages two, four, and five, and their differentiation capacity showed an irregular profile throughout the five culture passages analyzed. AT-MSCs showed a normal phenotype and differentiation capacity in all the culture passages. BM- and AT-MSCs did not modify their secretion ability or ultrastructural morphology. Conclusions: Throughout the culture passages, BM-MSCs, but not AT-MSCs, exhibited changes in their functional and phenotypic characteristic that might affect their efficacy as a cell therapy treatment. Therefore, the culture passage selected should be considered for the application of MSCs as a cell therapy treatment.

Cold Atmospheric Pressure Plasma-Activated Medium Modulates Cellular Functions of Human Mesenchymal Stem/Stromal Cells In Vitro.

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

Co-operation of MCL-1 and BCL-XL anti-apoptotic proteins in stromal protection of MM cells from carfilzomib mediated cytotoxicity.

Introduction: BCL-2 family proteins are important for tumour cell survival and drug resistance in multiple myeloma (MM). Although proteasome inhibitors are effective anti-myeloma drugs, some patients are resistant and almost all eventually relapse. We examined the function of BCL-2 family proteins in stromal-mediated resistance to carfilzomib-induced cytotoxicity in MM cells. Methods: Co-cultures employing HS5 stromal cells were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1-hour pulse method. The expression of BCL-2 family proteins was assessed by flow cytometry and WB. Pro-survival proteins: MCL-1, BCL-2 and BCL-XL were inhibited using S63845, ABT-199 and A-1331852 respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB. Results: CFZ induced dose-dependent cell death of MM cells, primarily mediated by apoptosis. Culture of MM cells on HS-5 stromal cells resulted in reduced cytotoxicity to CFZ in a cell contact-dependent manner, upregulated expression of MCL-1 and increased dependency on BCL-XL. Inhibiting BCL-XL or MCL-1 with BH-3 mimetics abrogated stromal-mediated protection only at high doses, which may not be achievable in vivo. However, combining BH-3 mimetics at sub-therapeutic doses, which alone were without effect, significantly enhanced CFZ-mediated cytotoxicity even in the presence of stroma. Furthermore, MCL-1 inhibition led to enhanced binding between BCL-XL and BIM, while blocking BCL-XL increased MCL-1/BIM complex formation, indicating the cooperative role of these proteins. Conclusion: Stromal interactions alter the dependence on BCL-2 family members, providing a rationale for dual inhibition to abrogate the protective effect of stroma and restore sensitivity to CFZ.

Optimizing cryopreservation conditions for use of fucosylated human mesenchymal stromal cells in anti-inflammatory/immunomodulatory therapeutics.

Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cell-based therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/anti-inflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics.

Optimization of the Amplification of Equine Muscle-Derived Mesenchymal Stromal Cells in a Hollow-Fiber Bioreactor.

The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m2. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull's eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications.