Current cell therapies for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which multiple organs are damaged by the immune system. Although standard treatment options such as hydroxychloroquine (HCQ), glucocorticoids (GCs), and other immunosuppressive or immune-modulating agents can help to manage symptoms, they do not offer a cure. Hence, there is an urgent need for the development of novel drugs and therapies. In recent decades, cell therapies have been used for the treatment of SLE with encouraging results. Hematopoietic stem cell transplantation, mesenchymal stem cells, regulatory T (Treg) cell, natural killer cells, and chimeric antigen receptor T (CAR T) cells are advanced cell therapies which have been developed and evaluated in clinical trials in humans. In clinical application, each of these approaches has shown advantages and disadvantages. In addition, further studies are necessary to conclusively establish the safety and efficacy of these therapies. This review provides a summary of recent clinical trials investigating cell therapies for SLE treatment, along with a discussion on the potential of other cell-based therapies. The factors influencing the selection of common cell therapies for individual patients are also highlighted.

Impact of tissue factor expression and administration routes on thrombosis development induced by mesenchymal stem/stromal cell infusions: re-evaluating the dogma.

BACKGROUND: Hyperactive coagulation might cause dangerous complications such as portal vein thrombosis and pulmonary embolism after mesenchymal stem/stromal cell (MSC) therapy. Tissue factor (TF), an initiator of the extrinsic coagulation pathway, has been suggested as a predictor of this process. METHODS: The expression of TF and other pro- and anticoagulant genes was analyzed in xeno- and serum-free manufactured MSCs. Furthermore, culture factors affecting its expression in MSCs were investigated. Finally, coagulation tests of fibrinogen, D-dimer, aPPTs, PTs, and TTs were measured in patient serum after umbilical cord (UC)-MSC infusions to challenge a potential connection between TF expression and MSC-induced coagulant activity.  RESULTS: Xeno- and serum-free cultured adipose tissue and UC-derived MSCs expressed the highest level of TF, followed by those from dental pulp, and the lowest expression was observed in MSCs of bone marrow origin. Environmental factors such as cell density, hypoxia, and inflammation impact TF expression, so in vitro analysis might fail to reflect their in vivo behaviors. MSCs also expressed heterogeneous levels of the coagulant factor COL1A1 and surface phosphatidylserine and anticoagulant factors TFPI and PTGIR. MSCs of diverse origins induced fibrin clots in healthy plasma that were partially suppressed by an anti-TF inhibitory monoclonal antibody. Furthermore, human umbilical vein endothelial cells exhibited coagulant activity in vitro despite their negative expression of TF and COL1A1. Patients receiving intravenous UC-MSC infusion exhibited a transient increase in D-dimer serum concentration, while this remained stable in the group with intrathecal infusion. There was no correlation between TF expression and D-dimer or other coagulation indicators. CONCLUSIONS: The study suggests that TF cannot be used as a solid biomarker to predict MSC-induced hypercoagulation. Local administration, prophylactic intervention with anticoagulation drugs, and monitoring of coagulation indicators are useful to prevent thrombogenic events in patients receiving MSCs. Trial registration NCT05292625. Registered March 23, 2022, retrospectively registered, https://www. CLINICALTRIALS: gov/ct2/show/NCT05292625?term=NCT05292625&draw=2&rank=1 . NCT04919135. Registered June 9, 2021, https://www. CLINICALTRIALS: gov/ct2/show/NCT04919135?term=NCT04919135&draw=2&rank=1 .

Obesity-associated epigenetic alterations and the obesity-breast cancer axis.

Both breast cancer and obesity can regulate epigenetic changes or be regulated by epigenetic changes. Due to the well-established link between obesity and an increased risk of developing breast cancer, understanding how obesity-mediated epigenetic changes affect breast cancer pathogenesis is critical. Researchers have described how obesity and breast cancer modulate the epigenome individually and synergistically. In this review, the epigenetic alterations that occur in obesity, including DNA methylation, histone, and chromatin modification, accelerated epigenetic age, carcinogenesis, metastasis, and tumor microenvironment modulation, are discussed. Delineating the relationship between obesity and epigenetic regulation is vital to furthering our understanding of breast cancer pathogenesis.

Perforated appendicitis due to fishbone.

Appendicitis is a common condition in daily clinical practice. Appendicitis due to foreign bodies is uncommon and may result from obstruction or perforation mechanism. We present a rare case of a 43-year-old male patient who was diagnosed with perforated appendicitis due to a fish bone by imaging studies and confirmed postoperatively. Confirming the fish bone causing the perforation on images is sometimes difficult, requiring the radiologist to actively search and determine the source. In addition to appendectomy, the surgeon also needs to pay attention to removing all foreign objects and treating perforations of surrounding organs.

Bone Marrow-Derived Mononuclear Cells in the Treatment of Neurological Diseases: Knowns and Unknowns.

Bone marrow-derived mononuclear cells (BMMNCs) have been used for decades in preclinical and clinical studies to treat various neurological diseases. However, there is still a knowledge gap in the understanding of the underlying mechanisms of BMMNCs in the treatment of neurological diseases. In addition, prerequisite factors for the efficacy of BMMNC administration, such as the optimal route, dose, and number of administrations, remain unclear. In this review, we discuss known and unknown aspects of BMMNCs, including the cell harvesting, administration route and dose; mechanisms of action; and their applications in neurological diseases, including stroke, cerebral palsy, spinal cord injury, traumatic brain injury, amyotrophic lateral sclerosis, autism spectrum disorder, and epilepsy. Furthermore, recommendations on indications for BMMNC administration and the advantages and limitations of BMMNC applications for neurological diseases are discussed. BMMNCs in the treatment of neurological diseases. BMMNCs have been applied in several neurological diseases. Proposed mechanisms for the action of BMMNCs include homing, differentiation and paracrine effects (angiogenesis, neuroprotection, and anti-inflammation). Further studies should be performed to determine the optimal cell dose and administration route, the roles of BMMNC subtypes, and the indications for the use of BMMNCs in neurological conditions with and without genetic abnormalities.

Factors associated with depression, anxiety, and stress symptoms among men in a rural area in Vietnam during COVID-19.

Background: The COVID-19 pandemic has affected health and well-being worldwide, and its psychological effects are receiving substantial attention in the scientific literature. Research to date shows that the pandemic has increased prevalences of depression, anxiety, and stress. This study aimed to estimate the prevalence of mental health symptoms and identify the associated factors among men in a rural area of Vietnam during the COVID-19 pandemic. Methods and findings: During July 15-31, 2020, we conducted a cross-sectional survey of 1,085 men from 18 years old in 11 rural districts in Thanh Hoa province, Vietnam, and assessed their mental health using the Depression, Anxiety and Stress Scale - 21 Items (DASS-21). Outcomes assessed were have a symptom of depression, anxiety, and stress; risk factors measured included age, religion, marital status, education, occupation, and financial status. Multiple linear regression was performed to determine the statistical significance of associations between risk factors and mental health symptoms. Findings showed that the prevalences of having a symptom of depression, anxiety and stress among participants were 6.39, 9.72, and 5.65%, respectively. Regression model showed being younger (95% CI: -0.030; -0.004, p = 0.001), men had high school degree (95% CI: -0.671; -0.074, p = 0.014), men living in nearly poor houshoulds (95% CI: 0.067, 1.905, p < 0.05) and poor housholds (95% CI: 0.608; 2.721, p < 0.05) had significantly lower depression scores than others. Conclusion: Prevalences of having symptoms of depression, anxiety and stress were much higher than in similar previous research in rural Vietnam, suggesting that mental health problems among men in this setting became more common during the COVID-19 pandemic. Age, religion, level of education and family income status were statistically significant predictors of mental health problems. These findings provide useful insights into the impact of pandemics on mental health.

"Adipose-derived mesenchymal stem cell therapy for the management of female sexual dysfunction: Literature reviews and study design of a clinical trial".

Hormone imbalance and female sexual dysfunction immensely affect perimenopausal female health and quality of life. Hormone therapy can improve female hormone deficiency, but long-term use increases the risk of cardiovascular diseases and cancer. Therefore, it is necessary to develop a novel effective treatment to achieve long-term improvement in female general and sexual health. This study reviewed factors affecting syndromes of female sexual dysfunction and its current therapy options. Next, the authors introduced research data on mesenchymal stromal cell/mesenchymal stem cell (MSC) therapy to treat female reproductive diseases, including Asherman's syndrome, premature ovarian failure/primary ovarian insufficiency, and vaginal atrophy. Among adult tissue-derived MSCs, adipose tissue-derived stem cells (ASCs) have emerged as the most potent therapeutic cell therapy due to their abundant presence in the stromal vascular fraction of fat, high proliferation capacity, superior immunomodulation, and strong secretion profile of regenerative factors. Potential mechanisms and side effects of ASCs for the treatment of female sexual dysfunction will be discussed. Our phase I clinical trial has demonstrated the safety of autologous ASC therapy for women and men with sexual hormone deficiency. We designed the first randomized controlled crossover phase II trial to investigate the safety and efficacy of autologous ASCs to treat female sexual dysfunction in perimenopausal women. Here, we introduce the rationale, trial design, and methodology of this clinical study. Because aging and metabolic diseases negatively impact the bioactivity of adult-derived MSCs, this study will use ASCs cultured in physiological oxygen tension (5%) to cope with these challenges. A total of 130 perimenopausal women with sexual dysfunction will receive two intravenous infusions of autologous ASCs in a crossover design. The aims of the proposed study are to evaluate 1) the safety of cell infusion based on the frequency and severity of adverse events/serious adverse events during infusion and follow-up and 2) improvements in female sexual function assessed by the Female Sexual Function Index (FSFI), the Utian Quality of Life Scale (UQOL), and the levels of follicle-stimulating hormone (FSH) and estradiol. In addition, cellular aging biomarkers, including plasminogen activator inhibitor-1 (PAI-1), p16 and p21 expression in T cells and the inflammatory cytokine profile, will also be characterized. Overall, this study will provide essential insights into the effects and potential mechanisms of ASC therapy for perimenopausal women with sexual dysfunction. It also suggests direction and design strategies for future research.

Stem cell-based therapy for human diseases.

Recent advancements in stem cell technology open a new door for patients suffering from diseases and disorders that have yet to be treated. Stem cell-based therapy, including human pluripotent stem cells (hPSCs) and multipotent mesenchymal stem cells (MSCs), has recently emerged as a key player in regenerative medicine. hPSCs are defined as self-renewable cell types conferring the ability to differentiate into various cellular phenotypes of the human body, including three germ layers. MSCs are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the International Society for Cell and Gene Therapy (ISCT). This review provides an update on recent clinical applications using either hPSCs or MSCs derived from bone marrow (BM), adipose tissue (AT), or the umbilical cord (UC) for the treatment of human diseases, including neurological disorders, pulmonary dysfunctions, metabolic/endocrine-related diseases, reproductive disorders, skin burns, and cardiovascular conditions. Moreover, we discuss our own clinical trial experiences on targeted therapies using MSCs in a clinical setting, and we propose and discuss the MSC tissue origin concept and how MSC origin may contribute to the role of MSCs in downstream applications, with the ultimate objective of facilitating translational research in regenerative medicine into clinical applications. The mechanisms discussed here support the proposed hypothesis that BM-MSCs are potentially good candidates for brain and spinal cord injury treatment, AT-MSCs are potentially good candidates for reproductive disorder treatment and skin regeneration, and UC-MSCs are potentially good candidates for pulmonary disease and acute respiratory distress syndrome treatment.

Human Umbilical Cord Mesenchymal Stem Cells for Severe Neurological Sequelae due to Anti-N-Methyl-d-Aspartate Receptor Encephalitis: First Case Report.

Anti-N-methyl-d-aspartate (NMDA) receptor encephalitis is caused by altered patient immune reactions. This study reports the first patient with severe neurologic sequelae after NMDA receptor encephalitis treated with allogeneic umbilical cord-derived mesenchymal stem/stromal cells (UC-MSCs). A 5-year-old girl was diagnosed with NMDA receptor encephalitis and treated with immunosuppressive medicaments and intravenous immunoglobulin (IVIG). Despite intensive therapy, the patient's condition worsened so that allogenic UC-MSC therapy was contemplated. The patient received three intrathecal infusions of xeno- and serum-free cultured UC-MSCs at a dose of 106 cells/kg. At baseline and after each UC-MSC administration, the patient was examined by the German Coma Recovery Scale (CRS), the Gross Motor Function Classification System (GMFCS), the Gross Motor Function Measure-88 (GMFM-88), the Manual Ability Classification System (MACS), the Modified Ashworth Scale, and the Denver II test. Before cell therapy, she was in a permanent vegetative state with diffuse cerebral atrophy. Her cognition and motor functions improved progressively after three UC-MSC infusions. At the last visit, she was capable of walking, writing, and counting numbers. Control of urinary and bowel functions was completely recovered. Cerebral atrophy was reduced on brain magnetic resonance imaging (MRI). Overall, the outcomes of this patient suggest a potential cell therapy for autoimmune encephalitis and its neurological consequences.

The role of MEK1/2 and MEK5 in melatonin-mediated actions on osteoblastogenesis, osteoclastogenesis, bone microarchitecture, biomechanics, and bone formation.

Melatonin, the primary hormone involved in circadian entrainment, plays a significant role in bone physiology. This study aimed to assess the role of MEK1/2 and MEK5 in melatonin-mediated actions in mouse and human mesenchymal stem cells (MSCs) and on bone using small-molecule inhibitors and CRISPR/Cas9 knockout approaches. Consistent with in vitro studies performed in mMSCs and hMSCs, nightly (25 mg/kg, i.p., 45 days) injections with PD184352 (MEK1/2 inhibitor) or Bix02189 (MEK5 inhibitor) or SC-1-151 (MEK1/2/5 inhibitor) demonstrated that MEK1/2 and MEK5 were the primary drivers underlying melatonin's actions on bone density, microarchitecture (i.e., trabecular number, separation, and connectivity density), and bone mechanical properties (i.e., ultimate stress) through increases in osteogenic (RUNX2, BMP-2, FRA-1, OPG) expression and decreases in PPARγ. Furthermore, CRISPR/Cas9 knockout of MEK1 or MEK5 in mMSCs seeded on PLGA scaffolds and placed into critical-size calvarial defects in Balb(c) mice (male and female) revealed that treatment with melatonin (15 mg/L; p.o., nightly, 90 days) mediates sex-specific actions of MEK1 and MEK5 in new bone formation. This study is the first to demonstrate a role for MEK1/2 and MEK5 in modulating melatonin-mediated actions on bone formation in vivo and in a sex-specific manner.

Clinical Study of Mesenchymal Stem/Stromal Cell Therapy for the Treatment of Frailty: A Proposed Experimental Design for Therapeutic and Mechanistic Investigation.

Frailty, a specific condition of increased vulnerability and reduced general health associated with aging in older people, is an emerging problem worldwide with major implications for clinical practice and public health. Recent preclinical and clinical studies have supported the safety of mesenchymal stem/stromal cells (MSCs) in the treatment of frailty. Comprehensive study is needed to assess the interrelationship between the condition of frailty and the effects of MSC-based therapy. This randomized controlled phase I/II trial aims to investigate the safety and potential therapeutic efficacy of the allogeneic administration of umbilical cord-derived MSCs (UC-MSCs) in combination with the standard treatment for frailty in Vietnam. Moreover, this study describes the rationales, study designs, methodologies, and analytical strategies currently employed in stem cell research and clinical studies. The primary outcome measures will include the incidences of prespecified administration-associated adverse events and serious adverse events. The potential efficacy will be evaluated based on improvements in frailty conditions (including those determined through a physical examination, patient-reported outcomes, quality of life, immune markers of frailty, metabolism analysis, and cytokine markers from patient plasma). This clinical trial and stem cell analysis associated with patient sampling at different time points aim to identify and characterize the potential effects of UC-MSCs on improving frailty based on the stem cell quality, cytokine/growth factor secretion profiles of UC-MSCs, cellular senescence, and metabolic analysis of patient CD3+ cells providing fundamental knowledge for designing and implementing research strategies in future studies. Clinical Trials Registration Number: NCT04919135.

Clinically relevant preservation conditions for mesenchymal stem/stromal cells derived from perinatal and adult tissue sources.

The interplay between mesenchymal stem/stromal cells (MSCs) and preservation conditions is critical to maintain the viability and functionality of these cells before administration. We observed that Ringer lactate (RL) maintained high viability of bone marrow-derived MSCs for up to 72 h at room temperature (18°C-22°C), whereas adipose-derived and umbilical cord-derived MSCs showed the highest viability for 72 h at a cold temperature (4°C-8°C). These cells maintained their adherence ability with an improved recovery rate and metabolic profiles (glycolysis and mitochondrial respiration) similar to those of freshly harvested cells. Growth factor and cytokine analyses revealed that the preserved cells released substantial amounts of leukaemia inhibitory factors (LIFs), hepatocyte growth factor (HGF) and vascular endothelial growth factor-A (VEGF-A), as well as multiple cytokines (eg IL-4, IL-6, IL-8, MPC-1 and TNF-α). Our data provide the simplest clinically relevant preservation conditions that maintain the viability, stemness and functionality of MSCs from perinatal and adult tissue sources.

Comparative Bioactivity Analysis for Off-the-Shelf and Culture-Rescued Umbilical Cord-Derived Mesenchymal Stem/Stromal Cells in a Xeno- and Serum-Free Culture System.

We recently reported a standardized xeno- and serum-free culture platform to isolate and expand umbilical cord-derived mesenchymal stem/stromal cells (UC-MSCs). Comparing populations from the same passage, cells that were cryopreserved and culture-rescued exhibited characteristics similar to those of their fresh counterparts, continuously cultured cells without interim cryopreservation. The culture rescue after thawing allowed for the cells to be fully recovered. However, since it would be more cost-effective and timesaving if cryopreserved cells can be used as an off-the-shelf product, we set out to compare the bioactivity of freshly thawed UC-MSCs versus culture-rescued UC-MSCs of the same batch that were recultured for an additional passage under our xeno- and serum-free protocol. UC-MSCs showed high viability in both the freshly thawed and the re-cultured group. Both populations displayed a similar proliferation capacity which is indicated by a comparable population doubling time and colony-forming ability. Both freshly thawed and culture-rescued UC-MSCs expressed the characteristic immunophenotype and were capable of differentiating into osteocytes, chondrocytes, and adipocytes. On the other hand, culture-rescued cells appeared to be more potent in immunosuppression than freshly thawed cells. In conclusion, freshly thawed and culture-rescued cell products share comparable bioactivity in cell growth and proliferation, immunophenotype, and differentiation potential. However, the culture-rescued cells that were allowed to grow for an additional passage appear to display a more favorable immunomodulatory potential when compared to their freshly thawed parent cells.

Human Adipose-Derived Mesenchymal Stromal Cells Exhibit High HLA-DR Levels and Altered Cellular Characteristics under a Xeno-free and Serum-free Condition.

BACKGROUND: We have observed an increased expression of negative markers in some clinical-grade, xeno- and serum-free cultured adipose-derived mesenchymal stem/stromal cell (ADMSC) samples. It gave rise to concern that xeno- and serum-free conditions might have unexpected effects on human ADMSCs. This study aims to test this hypothesis for two xeno- and serum-free media, PowerStem MSC1 media (PS) and StemMACS MSC Expansion Media (SM), that support the in vitro expansion of ADMSCs. METHODS: We investigated the expression of negative markers in 42 clinical-grade ADMSC samples expanded in PS. Next, we cultured ADMSCs from seven donors in PS and SM and examined their growth and colony-forming ability, surface marker expression, differentiation, cell cycle and senescence, as well as genetic stability of two passages representing an early and late passage for therapeutic MSCs. RESULTS: 15 of 42 clinical-grade PS-expanded ADMSC samples showed an increased expression of negative markers ranging from 2.73% to 34.24%, which positively correlated with the age of donors. This rise of negative markers was related to an upregulation of Human Leukocyte Antigen - DR (HLA-DR). In addition, the PS-cultured cells presented decreased growth ability, lower frequencies of cells in S/G2/M phases, and increased ß-galactosidase activity in passage 7 suggesting their senescent feature compared to those grown in SM. Although MSCs of both PS and SM cultures were capable of multilineage differentiation, the PS-cultured cells demonstrated chromosomal abnormalities in passage 7 compared to the normal karyotype of their SM counterparts. CONCLUSIONS: These findings suggest that the SM media is more suitable for the expansion of therapeutic ADMSCs than PS. The study also hints a change of ADMSC features at more advanced passages and with increased donor's age. Thus, it emphasizes the necessity to cover these aspects in the quality control of therapeutic MSC products.

Type 2 diabetes mellitus duration and obesity alter the efficacy of autologously transplanted bone marrow-derived mesenchymal stem/stromal cells.

Human bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) represent promising stem cell therapy for the treatment of type 2 diabetes mellitus (T2DM), but the results of autologous BM-MSC administration in T2DM patients are contradictory. The purpose of this study was to test the hypothesis that autologous BM-MSC administration in T2DM patient is safe and that the efficacy of the treatment is dependant on the quality of the autologous BM-MSC population and administration routes. T2DM patients were enrolled, randomly assigned (1:1) by a computer-based system into the intravenous and dorsal pancreatic arterial groups. The safety was assessed in all the treated patients, and the efficacy was evaluated based on the absolute changes in the hemoglobin A1c, fasting blood glucose, and C-peptide levels throughout the 12-month follow-up. Our data indicated that autologous BM-MSC administration was well tolerated in 30 T2DM patients. Short-term therapeutic effects were observed in patients with T2DM duration of <10 years and a body mass index <23, which is in line with the phenotypic analysis of the autologous BM-MSC population. T2DM duration directly altered the proliferation rate of BM-MSCs, abrogated the glycolysis and mitochondria respiration of BM-MSCs, and induced the accumulation of mitochondria DNA mutation. Our data suggest that autologous administration of BM-MSCs in the treatment of T2DM should be performed in patients with T2DM duration <10 years and no obesity. Prior to further confirming the effects of T2DM on BM-MSC biology, future work with a larger cohort focusing on patients with different T2DM history is needed to understand the mechanism underlying our observation.

Constitutive activation of MEK5 promotes a mesenchymal and migratory cell phenotype in triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited targeted therapeutic options. A defining feature of TNBC is the propensity to metastasize and acquire resistance to cytotoxic agents. Mitogen activated protein kinase (MAPK) and extracellular regulated kinase (ERK) signaling pathways have integral roles in cancer development and progression. While MEK5/ERK5 signaling drives mesenchymal and migratory cell phenotypes in breast cancer, the specific mechanisms underlying these actions remain under-characterized. To elucidate the mechanisms through which MEK5 regulates the mesenchymal and migratory phenotype, we generated stably transfected constitutively active MEK5 (MEK5-ca) TNBC cells. Downstream signaling pathways and candidate targets of MEK5-ca cells were based on RNA sequencing and confirmed using qPCR and Western blot analyses. MEK5 activation drove a mesenchymal cell phenotype independent of cell proliferation effects. Transwell migration assays demonstrated MEK5 activation significantly increased breast cancer cell migration. In this study, we provide supporting evidence that MEK5 functions through FRA-1 to regulate the mesenchymal and migratory phenotype in TNBC.

Dual inhibition of MEK1/2 and MEK5 suppresses the EMT/migration axis in triple-negative breast cancer through FRA-1 regulation.

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). Although MAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration (FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.

Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells.

Chemokine receptor 4 (CXCR4) and its ligand stromal-derived factor 1 (SDF-1) have well-characterized functions in cancer metastasis; however, the specific mechanisms through which CXCR4 promotes a metastatic and drug-resistant phenotype remain widely unknown. The aim of the present study was to demonstrate the application of a phenotypic screening approach using a small molecule inhibitor library to identify potential CXCR4-mediated signaling pathways. The present study demonstrated a new application of the Published Kinase Inhibitor Set (PKIS), a library of small molecule inhibitors from diverse chemotype series with varying levels of selectivity, in a phenotypic medium-throughput screen to identify potential mechanisms to pursue. Crystal violet staining and brightfield microscopy were employed to evaluate relative cell survival and changes to cell morphology in the screens. 'Hits' or lead active compounds in the first screen were PKIS inhibitors that reversed mesenchymal morphologies in CXCR4-activated breast cancer cells without the COOH-terminal domain (MCF-7-CXCR4-ΔCTD) and in the phenotypically mesenchymal triple-negative breast cancer cells (MDA-MB-231, BT-549 and MDA-MB-157), used as positive controls. In a following screen, the phenotypic and cell viability screen was used with a positive control that was both morphologically mesenchymal and had acquired fulvestrant resistance. Compounds within the same chemotype series were identified that exhibited biological activity in the screens, the 'active' inhibitors, were compared with inactive compounds. Relative kinase activity was obtained using published datasets to discover candidate kinase targets responsible for CXCR4 activity. MAP4K4 and MINK reversed both the mesenchymal and drug-resistant phenotypes, NEK9 and DYRK2 only reversed the mesenchymal morphology, and kinases, including ROS, LCK, HCK and LTK, altered the fulvestrant-resistant phenotype. Oligoarray experiments revealed pathways affected in CXCR4-activated cells, and these pathways were compared with the present screening approach to validate our screening tool. The oligoarray approach identified the integrin-mediated, ephrin B-related, RhoA, RAC1 and ErbB signaling pathways to be upregulated in MCF-7-CXCR4-ΔCTD cells, with ephrin B signaling also identified in the PKIS phenotypic screen. The present screening tool may be used to discover potential mechanisms of targeted signaling pathways in solid cancers.

Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources.

BACKGROUND AIMS: Mesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency. METHODS: The authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics. RESULTS: MSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications. CONCLUSIONS: The authors' data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products.