Factors In Conditioned Medium Research Articles

Periostin Is a Candidate Regulator of the Host Microenvironment in Regeneration of Pulp and Dentin Complex and Periodontal Ligament in Transplantation with Stem Cell-Conditioned Medium.

The microenvironment is required for tissues to maintain their properties in vivo. This microenvironment encompasses the types and three-dimensional arrangement of cells forming the tissues, and their interactions with neighboring cells and extracellular matrices, as represented by the stem cell niche. Tissue regeneration depends not on the original tissue source of the transplanted cells, but on the microenvironment in which they are transplanted. We have previously reported pulp regeneration in a heterotopic root graft model by transplantation of conditioned medium alone, which suggests that host-derived cells expressing receptors for migration factors in conditioned medium migrate into the root canal and cause pulp regeneration. Regenerative medicine is needed to restore the original function of complex tissues. To achieve this, it is necessary to reproduce the changes in the microenvironment of the host tissue that accompany the regenerative response. Therefore, it is important to reproduce the microenvironment in vivo for further development of tissue regeneration therapy. Periostin is also found in the epithelial-mesenchymal junction, with expression sites that differ depending on the mineralized matrix stage, and is involved in regulation of calcification. We investigate whether periostin contributes to microenvironmental changes in regenerated pulp tissue. Dental pulp stem cells were induced into dentin, and gene expression of DSPP, nestin, DMP1, Runx2, and periostin was analyzed by qPCR and protein expression by IHC. Similarly, gene expression was analyzed using qPCR and protein expression using IHC in regenerated dental pulp obtained by ectopic transplantation. Since these regenerated tissues were observable on the same slice, it was possible to understand changes in the microenvironment within the tissues. Periostin promoted proliferation of pulp stem cells, migration in type I collagen, and calcification in regenerated pulp, which strongly suggests that periostin is a promising candidate as a factor that contributes to the microenvironment of regenerated pulp.

Peritumoral adipose tissue promotes lipolysis and white adipocytes browning by paracrine action.

Stromal adipocytes and tumor breast epithelial cells undergo a mutual metabolic adaptation within tumor microenvironment. Therefore, browning and lipolysis occur in cancer associated adipocytes (CAA). However, the paracrine effects of CAA on lipid metabolism and microenvironment remodeling remain poorly understood. To analyze these changes, we evaluated the effects of factors in conditioned media (CM) derived from explants of human breast adipose tissue from tumor (hATT) or normal (hATN) on morphology, degree of browning, the levels of adiposity, maturity, and lipolytic-related markers in 3T3-L1 white adipocytes by Western blot, indirect immunofluorescence and lipolytic assay. We analyzed subcellular localization of UCP1, perilipin 1 (Plin1), HSL and ATGL in adipocytes incubated with different CM by indirect immunofluorescence. Additionally, we evaluated changes in adipocyte intracellular signal pathways. We found that adipocytes incubated with hATT-CM displayed characteristics that morphologically resembled beige/brown adipocytes with smaller cell size and higher number of small and micro lipid droplets (LDs), with less triglyceride content. Both, hATT-CM and hATN-CM, increased Pref-1, C/EBPβ LIP/LAP ratio, PPARγ, and caveolin 1 expression in white adipocytes. UCP1, PGC1α and TOMM20 increased only in adipocytes that were treated with hATT-CM. Also, hATT-CM increased the levels of Plin1 and HSL, while decreased ATGL. hATT-CM modified the subcellular localization of the lipolytic markers, favoring their relative content around micro-LDs and induced Plin1 segregation. Furthermore, the levels of p-HSL, p-ERK and p-AKT increased in white adipocytes after incubation with hATT-CM. In summary, these findings allow us to conclude that adipocytes attached to the tumor could induce white adipocyte browning and increase lipolysis as a means for endocrine/paracrine signaling. Thus, adipocytes from the tumor microenvironment exhibit an activated phenotype that could have been induced not only by secreted soluble factors from tumor cells but also by paracrine action from other adipocytes present in this microenvironment, suggesting a "domino effect".

An altered transcriptome underlies cln5-deficiency phenotypes in Dictyostelium discoideum.

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. The NCLs, commonly referred to as Batten disease, are a family of neurodegenerative lysosomal storage diseases that affect all ages and ethnicities globally. Previous research showed that CLN5 participates in a variety of cellular processes. However, the precise function of CLN5 in the cell and the pathway(s) regulating its function are not well understood. In the model organism Dictyostelium discoideum, loss of the CLN5 homolog, cln5, impacts various cellular and developmental processes including cell proliferation, cytokinesis, aggregation, cell adhesion, and terminal differentiation. In this study, we used comparative transcriptomics to identify differentially expressed genes underlying cln5-deficiency phenotypes during growth and the early stages of multicellular development. During growth, genes associated with protein ubiquitination/deubiquitination, cell cycle progression, and proteasomal degradation were affected, while genes linked to protein and carbohydrate catabolism were affected during early development. We followed up this analysis by showing that loss of cln5 alters the intracellular and extracellular amounts of proliferation repressors during growth and increases the extracellular amount of conditioned medium factor, which regulates cAMP signalling during the early stages of development. Additionally, cln5 - cells displayed increased intracellular and extracellular amounts of discoidin, which is involved in cell-substrate adhesion and migration. Previous work in mammalian models reported altered lysosomal enzyme activity due to mutation or loss of CLN5. Here, we detected altered intracellular activities of various carbohydrate enzymes and cathepsins during cln5 - growth and starvation. Notably, cln5 - cells displayed reduced β-hexosaminidase activity, which aligns with previous work showing that D. discoideum Cln5 and human CLN5 can cleave the substrate acted upon by β-hexosaminidase. Finally, consistent with the differential expression of genes associated with proteasomal degradation in cln5 - cells, we also observed elevated amounts of a proteasome subunit and reduced proteasome 20S activity during cln5 - growth and starvation. Overall, this study reveals the impact of cln5-deficiency on gene expression in D. discoideum, provides insight on the genes and proteins that play a role in regulating Cln5-dependent processes, and sheds light on the molecular mechanisms underlying CLN5 disease.

S1PR1 induces metabolic reprogramming of ceramide in vascular endothelial cells, affecting hepatocellular carcinoma angiogenesis and progression

Angiogenesis is a fundamental process underlying the occurrence, growth and metastasis of hepatocellular carcinoma (HCC), a prevalent tumour type with an extremely poor prognosis due to abundant vasculature. However, the underlying mechanism of angiogenesis in HCC remains largely unknown. Herein, we found that sphingosine-1-phosphate receptor 1 (S1PR1) plays an important role in HCC angiogenesis. S1PR1 was found to be selectively and highly expressed in the blood vessels of HCC tissues compared with those of paratumour tissues. Functionally, high expression of S1PR1 in endothelial cells (ECs) promoted angiogenesis and progression of HCC in vitro and in vivo. Mechanistically, proangiogenic factors (S1P, IL-6, VEGFA) in conditioned medium from HCC cells induced the upregulation of S1PR1 in ECs via the phosphorylation of STAT3 at Y705. Further study also revealed that S1PR1 promotes angiogenesis by decreasing ceramide levels via CerS3 downregulation. Interestingly, we demonstrated that S1PR1 downregulates CerS3 by inducing CerS6 translocation into the nucleus to inhibit CerS3 at the transcriptional level in ECs. In addition, we found that a high concentration of Lenvatinib significantly downregulated the expression of S1PR1 and obviously enhanced S1PR1 knockdown-mediated angiogenesis inhibition, indicating that S1PR1 may be a target by which Lenvatinib combats angiogenesis in HCC. Thus, S1PR1 may be an important target for suppressing angiogenesis in HCC, and inhibiting S1PR1 is a promising approach to antitumor therapy in HCC.

A study on the role of eugenol encapsulated liposomes in facilitating neuron -microglia mediated wound recovery

Activated microglia helps in resolving injury and supports wound healing. However, prolonged activation of microglia can lead to the aggravation of wound. ObjectiveThe present study investigates neuronal injury-induced inflammatory response in microglia and how it is influenced by the treatment with an anti-inflammatory formulation, Eugenol encapsulated liposomes. MethodWe introduced injury in an in vitro model of neuronal and microglia origin cell lines using scratch assay and evaluated wound closure rates with respect to treatment. On similar lines, both cell lines were treated in a combinatorial model using Eugenol encapsulated liposomes and investigated the effect of secreted factors in Conditioned Media (CM) on Reactive Oxygen Species (ROS) production, cell viability etc. We also evaluated the polarization properties of microglia in case of an injury by evaluating the amount of nitrite release. Further, in a co-culture model, we introduced neuronal injury in the vicinity of microglia. We then evaluated the inflammatory response of microglia after incubating the injured cells with the formulation for an hour. ResultsIndividual wounds, CM and co-culture models of neuronal injury treated with Eugenol encapsulated liposomes indicated a decrease in the production of ROS and pro-inflammatory microglia, with the latter displaying an enhanced difference (50–75% with respect to control). The markers produced by microglia cells with respect to an inflammatory condition followed by treatment with Eugenol encapsulated liposomes were investigated using ELISA and Western blotting. There was a reduction in the amount of pro-inflammatory markers TNF-α, IL-1β and an increase in the amount of anti-inflammatory markers IL-10 and IL-4. In control and treatment groups, cardiac rhythm was found to be in the basal levels (100–150 beats per minute). Viability of the embryos were also unaffected due to the treatment in individual group of 20 embryos each. Further, uptake of the formulation in embryos 2,3 & 4 days post fertilization (dpf) were analyzed. Co-encapsulating tracker dye, Nile red within the formulation indicated encapsulated tracker dye crossing organ barriers with respect to free dye. ConclusionNeuronal injury causes microglial activation. Prolonged microglial activation hinders wound closure and can lead to secondary trauma. The present study was conducted using an in vitro scratch assay wound model. Treating the site of injury with an anti-inflammatory formulation, Eugenol loaded liposomes, converts pro-inflammatory microglia to an anti-inflammatory state and increases the rate of wound closure.

P52 signaling promotes cellular senescence

BackgroundNuclear factor-κB is a multi-subunit transcription factor that plays a central role in cellular senescence. We previously reported that an increase in the p52 subunit is seen in senescent cells and aged tissue. In the current work, we examined the mechanism by which p52 is activated and whether the increase in p52 promotes senescence.ResultsUsing both primary mouse embryonic fibroblasts (MEFs) and WI-38 human lung fibroblasts, we examined cells after serial passage and following prolonged culture. An increase in p52 was found in the nucleus relative to pre-senescent cells. The increase in p52 protein was not reflected by an increase in NFKB2 mRNA or by an increase in the abundance of upstream activating kinases, IKKα and NIK. To examine whether p52 promotes senescence, we over-expressed mature p52 in primary MEFs. Significantly more senescence was seen compared to control, a finding not seen with p52 mutated at critical DNA binding residues. In addition, blocking p52 nuclear translocation with the peptide inhibitor, SN52, decreased β-galactosidase (β-gal) formation. Subsequent filtration studies demonstrated that proteins in conditioned media (CM) were necessary for the increase in p52 and mass spectrometry identified S100A4 and cyclophilin A (CYPA) as potential factors in CM necessary for induction of p52. The requirement of these proteins in CM for induction of p52 was confirmed using depletion and supplementation studies. In addition, we found that activation of STAT3 signaling was required for the increase in p52. Finally, genome wide ChIP-sequencing analysis confirmed that there is an increase in p52 chromatin enrichment with senescence and identified several downstream factors whose expression is regulated by increased p52 binding.ConclusionsThese results demonstrate that p52 nuclear translocation is increased in senescent cells by factors in conditioned media and that mature p52 induces cellular senescence. The data are consistent with the prior observation that p52 is elevated in aged tissue and support the hypothesis that p52 contributes to organismal aging.

Endothelial Cell Tube Formation Assay: An In Vitro Model for Angiogenesis.

Endothelial cell tube formation assay is one of the most widely used and reliable methods for studying in vitro angiogenesis. Endothelial cells plated over a basement membrane extract and subjected to angiogenic factors in conditioned medium, form a rapid and quantifiable tube network within hours. Tube formation is sustained for 18-24h, after which time apoptosis occurs and tube networks disintegrate. The tube network can be imaged using a phase contrast microscope, or upon Calcein-AM treatment, a fluorescence/confocal microscope. This assay has several advantages, namely: ease of set up, the ability to test numerous angiogenic/anti-angiogenic factors simultaneously, quick network formation, ability to view live or fixed tube networks, and quantifiability. To ensure successful results and limit variability, proper selection of basement membrane extracts and endothelial cells is necessary, and conditions must be optimized. In summary, this assay is a useful method for screening potential angiogenic/anti-angiogenic factors as well as identifying critical mechanisms and signaling pathways underlying angiogenic-related pathologies.

Regulatory roles of miRNAs 16, 133a, and 223 on osteoclastic bone destruction caused by breast cancer metastasis.

Osteolytic bone metastasis leads to skeletal-related events, resulting in a decline in the patient activities and survival; therefore, it is important to understand the mechanism underlying bone metastasis. Recent studies have suggested that microRNAs (miRNAs or miRs) are involved in osteoclast differentiation and/or osteolytic bone metastasis; however, the roles of miRNAs have not been elucidated. In the present study, the roles of miRNAs in bone destruction caused by breast cancer metastasis were investigated in vitro and in vivo. miR-16, miR-133a and miR-223 were transfected into a human breast cancer cell line, MDA-MB-231. The expression of osteolytic factors in conditioned medium (miR-CM) collected from the culture of transfected cells was assessed. To evaluate the effects of miRNAs on osteoclast differentiation and activities, tartrate-resistant acid phosphatase (TRAP) staining and bone resorptive assays were performed in osteoclasts following miR-CM treatment. To create in vivo bone metastasis models for histological and morphometric evaluation, miRNA-transfected MDA-MB-231 cells were transplanted into the proximal tibia of nude mice. Expression of osteolytic factors, including receptor activator for nuclear factor-κB ligand (RANKL), interleukin (IL)-1β, IL-6, parathyroid hormone-related protein (PTHrP), and tumor necrosis factor (TNF), was increased in miR-16-CM, whereas it was decreased in both miR-133a-CM and miR-223-CM. TRAP staining and bone resorptive assays revealed that osteoclast function and activities were promoted by miR-16-CM treatment, whereas they were suppressed by miR-133a-CM and miR-223-CM. Consistent with in vitro findings, in vivo experiments revealed that the overexpression of miR-16 increased osteoclast activities and bone destruction in MDA-MB-231 cells, whereas the opposite results were observed in both miR-133a- and miR-223-transfected MDA-MB-231 cells. Our results indicated that miR-16 promoted osteoclast activities and bone destruction caused by breast cancer metastasis in the bone microenvironment, whereas miR-133a and miR-223 suppressed them. These miRNAs could be potential biomarkers and therapeutic targets for breast cancer bone metastasis.

APOE4-carrying human astrocytes oversupply cholesterol to promote neuronal lipid raft expansion and Aβ generation.

SummaryThe ε4 allele of APOE-encoding apolipoprotein (ApoE) is one of the strongest genetic risk factors for Alzheimer’s disease (AD). One of the overarching questions is whether and how this astrocyte-enriched risk factor initiates AD-associated pathology in neurons such as amyloid-β (Aβ) accumulation. Here, we generate neurons and astrocytes from isogenic human induced pluripotent stem cells (hiPSCs) carrying either APOE ε3 or APOE ε4 allele and investigate the effect of astrocytic ApoE4 on neuronal Aβ production. Secretory factors in conditioned media from ApoE4 astrocytes significantly increased amyloid precursor protein (APP) levels and Aβ secretion in neurons. We further found that increased cholesterol secretion from ApoE4 astrocytes was necessary and sufficient to induce the formation of lipid rafts that potentially provide a physical platform for APP localization and facilitate its processing. Our study reveals the contribution of ApoE4 astrocytes to amyloidosis in neurons by expanding lipid rafts and facilitating Aβ production through an oversupply of cholesterol.

ВЛИЯНИЕ МЕЗЕНХИМАЛЬНЫХ СТВОЛОВЫХ КЛЕТОК ЖИРОВОЙ ТКАНИ И ПАРАКРИННЫХ ФАКТОРОВ КОНДИЦИОННОЙ СРЕДЫ НА ЗАЖИВЛЕНИЕ ЛУЧЕВЫХ ЯЗВ ПРИ ТЕРАПИИ ТЯЖЕЛЫХ РАДИАЦИОННЫХ ПОРАЖЕНИЙ КОЖИ У КРЫС

Purpose: Studying of the effects transplantation of cultured mesenchymal stem cells of adipose tissue (MMSC) and adipose-derived stromal vascular fraction (SVF), as well as the introduction of paracrine factors (PF) of conditioned medium in an isolated or combined application for severe local radiation skin lesions in the experiment.
 Material and methods: Rats of the inbred Wistar–Kyoto strain were irradiated to local X-rays exposure in the iliolumbar region of the back at a dose of 110 Gy. The transplantation of cultured MMSC was performed twice at doses of 2.1 × 106 and 2.6 × 106 on the 28th and 35th days after irradiation. Adipose-derived SVF was administered at the same time in doses of 3.2 × 106 and 2.8 × 106, respectively. PF were administered five times from the 1st to the 10th day after irradiation. The severity of radiation damage to the skin and the effects of therapy were evaluated in dynamics by clinical manifestations, using planimetry and histological methods.
 Results: Radiation exposure with these parameters caused severe radiation injuries of the skin with non-healing ulcers formed by the 21–25th day after irradiation. The area of radiation ulcers in rats of the control group in the period from the 26th to the 83rd day slowly decreased from 2.76 ± 0.12 cm2 to 1.85 ± 0.13 cm2. In 50 % of the animals in the control group, ulcers persisted for more than 4 months after irradiation. In rats of the experimental groups, more intensive healing and a decrease in the area of radiation ulcers was noted. With isolated administration of cultured MMSC or SVF, a statistically significant decrease in the area of ulcers compared with the control was observed on the 104–125th day, and with the introduction of PF on the 83rd day after irradiation, p <0.05. In the control group, by the118th day after irradiation, radiation ulcers healed only in 25 % of rats, and in the experimental groups with isolated administration of cultured MMSC, SVF and PF in 40–55 % of the rats showed complete epithelialization of wounds with the formation of an atrophic scar. Under the conditions of combined use of stem cells and conditioned medium factors, the number of animals with complete healing of radiation ulcers was 85–100 % by 118th days, p <0.05.
 Conclusion: Thus, transplantation of cultured MMSC of adipose tissue and adipose-derived SVF, as well as the introduction of PF of conditioned medium, can enhance the regeneration processes and stimulate skin regeneration, promoting earlier healing of chronic radiation ulcers in severe local radiation injuries. Moreover, with the combined introduction of PF and adipose-derived stem cell transplantation, the effectiveness of the healing of radiation ulcers was increases.

Stem cell secretome, regeneration, and clinical translation: a narrative review.

Regenerative medicine is a field growing in popularity due to high hopes for stimulating in situ tissue restoration. Stem cell therapy remain at the center of regenerative medicine, due to early reports on its pluripotent differentiating capability. However, more recent reports suggest the paracrine activity of stem cells, and not direct differentiation, as the cause of its therapeutic effects. This paracrine activity can be harnessed in the form of conditioned media. Despite these capabilities, the clinical translation of stem cell conditioned media (i.e., secretome) is precluded by a variety of factors. These limitations include standardization of stem cell-conditioned media formulation, characterization of bioactive factors in conditioned media and dosing, optimizing modes of delivery, and uncovering of mechanisms of action of stem cell conditioned media. The purpose of this review is to provide a focused narration on the aforementioned preclusions pertaining to the clinical translation of stem cell conditioned media. Specifically, we will report on commonly use methodologies for the development of stem cell conditioned media, modalities for conditioned media characterization, modes of delivery, and postulated mechanisms of action for stem cell conditioned media in regenerative medicine.

Knockdown of EMMPRIN (OX47) in MRMT-1 Carcinoma Cells Inhibits Tumor Growth and Decreases Cancer-Induced Bone Destruction and Pain.

PurposeBone destruction and pain caused by cancer is one of the most devastating complications of cancer patients with bone metastases, and it seriously affects the quality of patients’ life. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell adhesion molecule with increased expression in a variety of tumors. This study focused to clarify the specific function of EMMPRIN in bone metastasis of breast cancer.Materials and MethodsAdenovirus with shRNA-EMMPRIN was transfected into MRMT-1 rat breast carcinoma cells, and the MRMT-1 cells with different expression levels of EMMPRIN were implanted into the bone marrow cavity of rat tibia. Next, the effect of down-regulation of EMMPRIN was evaluated as follows: bone damage was detected by X-ray radiological and tartrate-resistant acid phosphatase staining; the tumor burden was evaluated by hematoxylin and eosin staining; the test of pain-related behaviors was assessed used the bilateral paw withdrawal mechanical threshold; and the levels of secretory factors in tumor conditioned medium were determined by using enzyme-linked immunosorbent assay.ResultsWe found that down-regulation of EMMPRIN in tumor cells can simultaneously reduce tumor burden, relieve cancer-induced bone destruction and pain.ConclusionEMMPRIN is expected to be a therapeutic target for relieving bone metastasis of breast cancer and alleviating cancer-induced bone destruction and pain. The method of targeting EMMPRIN may be a promising strategy for the treatment of cancer in the future.

Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects

Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects

Cellular velocity, electrical persistence and sensing in developed and vegetative cells during electrotaxis.

Cells have the ability to detect electric fields and respond to them with directed migratory movement. Investigations identified genes and proteins that play important roles in defining the migration efficiency. Nevertheless, the sensing and transduction mechanisms underlying directed cell migration are still under discussion. We use Dictyostelium discoideum cells as model system for studying eukaryotic cell migration in DC electric fields. We have defined the temporal electric persistence to characterize the memory that cells have in a varying electric field. In addition to imposing a directional bias, we observed that the electric field influences the cellular kinematics by accelerating the movement of cells along their paths. Moreover, the study of vegetative and briefly starved cells provided insight into the electrical sensing of cells. We found evidence that conditioned medium of starved cells was able to trigger the electrical sensing of vegetative cells that would otherwise not orient themselves in the electric field. This observation may be explained by the presence of the conditioned medium factor (CMF), a protein secreted by the cells, when they begin to starve. The results of this study give new insights into understanding the mechanism that triggers the electrical sensing and transduces the external stimulus into directed cell migration. Finally, the observed increased mobility of cells over time in an electric field could offer a novel perspective towards wound healing assays.

Mini-Review; Deriving Avian Stem Cells by Small Molecules.

Avian embryos and related cell lines have found wide applications in basic and applied sciences. The embryonated egg is a great host for monoclonal antibodies and recombinant proteins. Avian cell lines derived from embryonated eggs have been used for the production of transgenic birds and virus inoculation in vaccine preparation. Hitherto, many efforts have been invested to develop efficient avian stem cell culture. Under the conventional conditions, there are various challenges, such as the type of feeder layers, conditioned medium, serum, and growth factors. Researchers have investigated different conditions to solve these problems. Recent studies have shown that targeted strategies using small molecule inhibitors could be used as alternatives to multi-growth factor delivery approaches. Since small molecule inhibitors were used for mammalian pluripotent stem cells (PSC), several kinds of research have examined the effect of the small molecule on self- -renewal and maintenance of avian PSC. Avian PSC can be derived from early blastodermal cells (stage X), circular primoridial germ cells (PGC; stage HH17), gonadal PGC (stage HH28), and embryonic germ cells (EGC; HH28). Previous studies have shown that the use of small molecule drugs such as PD0325901, SB431542, SC1, IDE1, Z-VAD, Blebbistatin, H-1152, and IDE1 could be an efficient method for the derivation of avian stem cells. This mini-review covers the recent development of avian stem cell culture by small molecules.

Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling.

Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)? SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization. Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis. This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n= 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization. eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs. SP promoted decidualization both in the absence and presence of steroid hormones (P< 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization. RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640. This study is limited to in vitro analyses. Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation. This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.

АДАПТАЦИЯ МЕТОДА ИНТЕРФЕРОМЕТРИИ СЛОЯ БИОМОЛЕКУЛ ДЛЯ КОЛИЧЕСТВЕННОЙ ОЦЕНКИ СОДЕРЖАНИЯ ФАКТОРА РОСТА ЭНДОТЕЛИЯ СОСУДОВ В КОНДИЦИОНИРОВАННОЙ КЛЕТОЧНОЙ СРЕДЕ

Quantitative analysis of cytokines, chemokines, growth factors and other soluble proteins in different biological liquids is routinely performed in contemporary diagnostics and biomedicine research. However, current methods of analysis are time-consuming and include multiple steps. In this study, we have developed a protocol that describes how bio-layer interferometry can be applied to quantify an analyte in several minutes. Conditioned growth media collected from mouse mesenchymal stem cells grown in normoxia or hypoxic conditions in a monolayer fashion, MSC-derived 3D cell sheets and 3D spheroids were used as a model system in which we determined a concentration of vascular endothelial growth factor (VEGF-A). This technique displayed a high sensitivity (down to 0.1 ng of VEGF-A per mL as a minimum). The measured concentrations of VEGF-A in the conditioned media from mesenchymal stem cells turned out to be similar with values determined by the enzyme-linked immunosorbent assay. Using bio-layer interferometry, it was shown that as compared to mesenchymal stem cells grown in monolayer, spheroids and 3D sheets of mesenchymal stem cells produce significantly more VEGF-A (by 2.5-3.0-fold). Thus, due to the developed protocol it was possible to adapt bio-layer interferometry for rapid quantification of growth factors in conditioned media.

Secreted factors from cultured dental pulp stem cells promoted neurite outgrowth of dorsal root ganglion neurons and ameliorated neural functions in streptozotocin-induced diabetic mice.

Aims/IntroductionTransplantation of stem cells promotes axonal regeneration and angiogenesis in a paracrine manner. In the present study, we examined whether the secreted factors in conditioned medium of stem cells from human exfoliated deciduous teeth (SHED‐CM) had beneficial effects on diabetic polyneuropathy in mice.Materials and MethodsConditioned medium of stem cells from human exfoliated deciduous teeth was collected 48 h after culturing in serum‐free Dulbecco's modified Eagle's medium (DMEM), and separated into four fractions according to molecular weight. Dorsal root ganglion neurons from C57BL/6J mice were cultured with SHED‐CM or DMEM to evaluate the effect on neurite outgrowth. Streptozotocin‐induced diabetic mice were injected with 100 μL of SHED‐CM or DMEM into the unilateral hindlimb muscles twice a week over a period of 4 weeks. Peripheral nerve functions were evaluated by the plantar test, and motor and sensory nerve conduction velocities. Intraepidermal nerve fiber densities, capillary number‐to‐muscle fiber ratio, capillary blood flow and morphometry of sural nerves were also evaluated.ResultsConditioned medium of stem cells from human exfoliated deciduous teeth significantly promoted neurite outgrowth of dorsal root ganglion neurons compared with DMEM. Among four fractions of SHED‐CM, the only fraction of <6 kDa promoted the neurite outgrowth of dorsal root ganglion neurons. In addition, SHED‐CM significantly prevented decline in sensory nerve conduction velocities compared with DMEM in diabetic mice. Although SHED‐CM did not improve intraepidermal nerve fiber densities or morphometry of sural nerves, SHED‐CM ameliorated the capillary number‐to‐muscle fiber ratio and capillary blood flow.ConclusionsThese results suggested that SHED‐CM might have a therapeutic effect on diabetic polyneuropathy through promoting neurite outgrowth, and the increase in capillaries might contribute to the improvement of neural function.

Human Umbilical Tissue-Derived Cells Secrete Soluble VEGFR1 and Inhibit Choroidal Neovascularization

Exudative age-related macular degeneration (AMD), characterized by choroidal neovascularization (CNV), is the leading cause of irreversible blindness in developed countries. Anti-vascular endothelial growth factor (VEGF) drugs are the standard treatment for AMD, but they have limitations. Cell therapy is a promising approach for ocular diseases, and it is being developed in the clinic for the treatment of retinal degeneration, including AMD. We previously showed that subretinal injection of human umbilical tissue-derived cells (hUTCs) in a rodent model of retinal degeneration preserved photoreceptors and visual function through rescue of retinal pigment epithelial (RPE) cell phagocytosis. Here we investigated the effect of hUTCs on a rat model of laser-induced CNV and on a human RPE cell line, ARPE-19, for VEGF production. We demonstrate that subretinal injection of hUTCs significantly inhibited CNV and lowered choroidal VEGF in vivo. VEGF release from ARPE-19 decreased when co-cultured with hUTCs. Soluble VEGF receptor 1 (sVEGFR1) is identified as the only factor in hUTC conditioned medium (CM) that binds to VEGF. The level of exogenous recombinant VEGF in hUTC CM was dramatically reduced and could be recovered with sVEGFR1-neutralizing antibody. This suggests that hUTC inhibits angiogenesis through the secretion of sVEGFR1 and could serve as a novel treatment for angiogenic ocular diseases, including AMD.

The impact of conditioned medium of umbilical cord-derived mesenchymal stem cells toward apoptosis and proliferation of glioblastoma multiforme cells

Glioblastoma multiforme (GBM) is the human malignant and highly invasive brain tumor. Tumor growth and invasive property are affected by mesenchymal stem cells (MSCs) as a part of tumor microenvironment. However, accumulating evidences describe that MSCs could act as pro and anti tumorigenic. The different action of MSCs in regulating tumor growth is mediated by secreted factor. This research purpose was to analyze the impact of MSCs secreted factor in conditioned medium (CM) on apoptosis and proliferation of GBM cells. Primary culture of umbilical cord-derived MSCs (UCSCs) was conducted in this research. CM-UCSCs was prepared by culturing the UCSCs in serum free aMEM for 24 hours. Those CM-UCSCs was 2-fold diluted by Dulbecco’s Modified Eagle Medium (DMEM) high glucose (50% concentration) and used to treat human GBM T98G cells for 24 hours. Following this treatment, apoptosis was detected using annexin V-FITC and cells proliferation was analyzed using trypan blue exclusion test. The results showed that early apoptosis occurred in 14.4 % of control cells and 14.8 % of CM-treated T98G cells, as well as late apoptosis occurred in 7.8 % of control cells and 7.2 % of CM-treated T98G cells. Whereas, GBM cells proliferation was tend to higher in the CM treated cells (3.85×105 cells) compared to the control (2.97×105 cells). In conclusion, CM-UCSCs does not seems to affect apoptosis, but tend to increase cells proliferation. Further research is needed to elaborate the mechanism of UCSCs secretome in stimulating cells proliferation.