Mesenchymal Cell Types Research Articles

Circular RNA CDR1as promotes adipogenic and suppresses osteogenic differentiation of BMSCs in steroid-induced osteonecrosis of the femoral head.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a common debilitating orthopedic disease. The bone marrow mesenchymal stem cells (BMSCs) are a type of mesenchymal stem cells which play crucial roles in bone repair. The adipogenic/osteogenic differentiation disorder of BMSCs has been widely perceived contributing to SONFH. However, the regulatory mechanism of BMSCs differentiation disorder still remains unclear. Circular RNA (circRNA), a kind of stable ncRNA, plays important roles in regulating gene expression via various ways. To date, there are no studies to uncover the circRNA expression profile and screen out the key circRNAs playing crucial roles in adipogenic/osteogenic differentiation disorder of SONFH-BMSCs. In present study, we detected the circRNA expression profiles in SONFH-BMSCs for the first time. A total of 820 circRNAs were differentially expressed in SONFH-BMSCs, including 460 up- and 360 down-regulated circRNAs. Bioinformatics analysis indicates circRNA CDR1as, one up-regulated circRNA, may play crucial role in adipogenic/osteogenic differentiation disorder of SONFH-BMSCs via CDR1as-miR-7-5p-WNT5B axis. Knocking-down CDR1as resulted in increasing of osteogenic differentiation and decreasing of adipogenic differentiation of BMSCs, while over-expressing CDR1as resulted in decreasing of osteogenic differentiation and increasing of adipogenic differentiation of BMSCs. The miR-7-5p binding sites of CDR1as and WNT5B were verified by luciferase reporter gene assay. Our study may provide new insights into the molecular mechanisms of osteogenic/adipogenic differentiation disorder of SONFH-BMSCs and new biomarkers for the diagnosis and treatment of SONFH.

Adipose-derived stem cells and cancer cells fuse to generate cancer stem cell-like cells with increased tumorigenicity.

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44+ CD24-/low EpCAM+ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.

PuraMatrix allows differentiation of a broad repertoire of neural and mesenchymal phenotypes from trunk neural crest.

The neural crest (NC) is a transitory embryonic structure of vertebrates that gives rise to an astonishing variety of derivatives, encompassing both neural and mesenchymal cell types. Neural crest cells (NCCs) are an excellent model to study how environmental factors modulate features such as cell multipotentiality and differentiation. Tests with multifunctional substrates that allow NCCs to express their full potential, while promoting cell subcloning, are needed to advance knowledge about NCC self-renewal and to foster future biotechnological approaches. Here we show that a self-assembled peptide named PuraMatrixTM is an excellent substrate that allows the differentiation of NCCs based on the identification of seven different cell types. Depending on the PuraMatrixTM concentration employed, different frequencies and quantities of a given cell type were obtained. It is noteworthy that an enormous quantity and diversity of mesenchymal phenotypes, such as chondrocytes, could be observed. The quantity of adipocytes and osteocytes also increased with the use of mesenchymal differentiation factors (MDF), but PuraMatrixTM alone can support the appearance of these mesenchymal cell types. PuraMatrixTM will promote advances in studies related to multipotentiality, self-renewal and control of NCC differentiation, since it is an extremely simple and versatile material which can be employed for both in vivo and in vitro experiments.

GMP-Compliant Production of Human Placenta-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells are one of the most attractive sources for stem cell research and therapy. Their safety and efficacy have been demonstrated in many clinical trials. Because of their low immunogenicity and immunomodulatory properties, allogenic MSCs have been transplanted in different clinical studies. MSCs could be in different adult- and fetal-derived tissues including pregnancy products. Placenta-derived mesenchymal stem cells (PLMSCs) that can be harvested without using any invasive procedures from a discarding tissue are one of the important types of mesenchymal stem cells for therapeutic applications. Stem cell manufacturing for therapeutic applications should be in compliance with the basic principles of good manufacturing practice (GMP). Herein, the current chapter is to describe GMP-compliant production of human PLMSCs, which are suitable for clinical applications.

Basal characterization and in vitro differentiation of putative stem cells derived from the adult mouse ovary

Lately, stem cell approaches have provided new information on reproductive organ function and additionally recommended novel treatment possibilities. The type(s) and differentiation potential of stem cells present in the mammalian ovary are largely unknown; while oogonial stem cells have been reported, we explored the possibility that multipotent stem cells may reside in the ovary and have wide differentiation potential. In this experimental study, homogenates of whole mouse ovaries were sorted using the stem cell surface markers stem cell antigen-1 and stage specific embryonic antigen-1/CD15. Viable double-positive cells 3-10μm in diameter were evaluated immediately after sorting and after culture using differentiation conditions. Ovarian-derived stem cells were differentiated into the three main cell types: adipocytes, chondrocytes, or osteocytes. The subsequent culture was performed in media containing bone morphogenetic protein 4 (BMP-4) and/or retinoic acid (RA). RA, BMP-4 or the two agents in combination, consistently stimulated germ cell gene expression. RA treatment strongly stimulated germline gene expression and also the development of cells that were morphologically reminiscent of oocytes. The germ cell genes Dazl, Ddx4, Figla, Gdf-9, Nobox, Prdm9, and Sycp-1 were all detected at low levels. Remarkably, treatment with BMP-4 alone significantly increased protein expression of the granulosa cell product anti-Müllerian hormone (AMH). We have shown that an inclusive isolation protocol results in the consistent derivation of multipotent stem cells from the adult ovary; these cells can be differentiated towards the germ cell fate (RA alone), somatic ovarian cell fate as indicated by AMH production (BMP-4 alone), or classical mesenchymal cell types. Taken together, these data suggest the presence of multipotent mesenchymal stem cells in the murine ovary.

Distinct interactions between epithelial and mesenchymal cells control cell morphology and collective migration during sponge epithelial to mesenchymal transition.

Epithelial and mesenchymal cell types are basic for animal multicellularity and they have complementary functions coordinated by cellular interactions. Sponges are especially important model organisms to address the evolutionary basis of morphogenetic programs for epithelial and mesenchymal organization in animals. Evolutionary studies in sponges can contribute to the understanding of the mechanisms that control tissue maintenance and tumor progression in humans. In the present study, sponge mesenchymal and epithelial cells were isolated from the demosponge Hymeniacidon heliophila, and aggregate formation was observed by video microscopy. Epithelial-mesenchymal interaction, epithelial transition, and cell migration led to sponge cell aggregation after drastic stress. Based on their different morphologies, adhesion specificities, and motilities, we suggest a role for different sponge cell types as well as complementary functions in cell aggregation. Micromanipulation under the microscope and cell tracking were also used to promote specific grafting-host interaction, to further test the effects of cell type interaction. The loss of cell polarity and flattened shape during the epithelial to mesenchymal cell transition generated small immobile aggregates of round/amoeboid cells. The motility of these transited epithelial-cell aggregates was observed by cell tracking using fluorescent dye, but only after interaction with streams of migratory mesenchymal cells. Cell motility occurred independently of morphological changes, indicating a progressive step in the transition toward a migratory mesenchymal state. Our data suggest a two-step signaling process: (a) the lack of interaction between mesenchymal and epithelial cells triggers morphological changes; and (b) migratory mesenchymal cells instruct epithelial cells for directional cell motility. These results could have an impact on the understanding of evolutionary aspects of metastatic cancer cells. HIGHLIGHTS: Morphogenetic movements observed in modern sponges could have a common evolutionary origin with collective cell migration of human metastatic cells. A sponge regenerative model was used here to characterize epithelial and mesenchymal cells, and for the promotion of grafting/host interactions with subsequent cell tracking. The transition from epithelial to mesenchymal cell type can be observed in sponges in two steps: (a) withdrawal of epithelial/mesenchymal cell interactions to trigger morphological changes; (b) migratory mesenchymal cells to induce epithelial cells to a collective migratory state.

Resveratrol Effect on Adipose-Derived Stem Cells Differentiation to Chondrocyte in Three-Dimensional Culture.

Purpose: Adipose stem cells (ASCs) are pluripotent cells with the ability of self-renewal and differentiation into different types of mesenchymal cells. As cartilage repair is difficult due to lack of blood capillary, resveratrol (Res) is a polyphenolic compound with diverse biological properties to be possibly used in this case. The aim of the present study was to investigate the effect of Res on differentiation of ASCs into chondrocyte in a three-dimensional (3D) culture model. Methods: Subcutaneous adipose tissues were prepared and digested enzymatically, and passed through cell strainer. ASCs were harvested in the fourth passage, and divided into five groups. The control group received chondrogenic differentiation medium (CDM) while the experimental groups received CDM plus different doses of Res (1, 10, 20, and 50 µM) for 21 days. Expression of cartilage specific genes and Sirtuin1 (SIRT 1), cell viability, apoptosis and ferric reducing antioxidant power (FRAP) were detected using reverse transcription polymerase chain reaction (RT-PCR), MTT assay, TUNEL and acridine orange/ethidium bromide (AO/EB) staining. One-way ANOVA and non-parametric Mann-Whitney U test were used for data analyses. Results: ASCs were differentiated to chondrocyte by CDM in a three-dimensional culture. 10 and 20 µM doses of Res showed the most proliferating effect on ADSCs. The SIRT 1 genes expression and FRAP level also increased significantly compared to the control group (P<0.05). Also, OD of cell increased whereas apoptosis decreased. Conclusion: 3D culture was a suitable condition for ASCs differentiation to chondrocyte, and lower doses of Res exert proliferation effect on ASCs.

FoxD1-driven CCN2 deletion causes axial skeletal deformities, pulmonary hypoplasia, and neonatal asphyctic death.

Pulmonary fibrosis is a severely disabling disease often leading to death. CCN2 (Cellular Communication Network factor 2, also known as CTGF) is a known mediator of fibrosis and clinical trials studying anti-CCN2 efficacy in pulmonary fibrosis are currently underway. Fork head box D1 (FoxD1) transcription factor is transiently expressed in several mesenchymal cell types, including those of fetal lungs. Differentiation of FoxD1-progenitor derived pericytes into myofibroblasts involves CCN2 expression and contributes importantly to maladaptive tissue remodeling in e.g. kidney and lung fibrosis models. To generate a model for studying the contribution of CCN2 expression in FoxD1-progenitor derived cells to development of fibrotic tissue remodeling, we set out to establish a FoxD1Cre - CCN2flox/flox mouse colony. However, all double-transgenic mice died soon after birth due to asphyxia. Histopathological examination revealed a reduction in alveolar space and lung weight, and subtle axial (thoracic and cervical) skeletal deformities. Together with the previously reported association of a FoxD1 containing locus with human adolescent idiopathic scoliosis, our data suggest that the development of fatal pulmonary hypoplasia caused by selective deletion of CCN2 from FoxD1-progenitor derived mesenchymal cells was secondary to aberrant axial skeletogenesis.

The Effect of Photobiomodulation Therapy on the Differentiation, Proliferation, and Migration of the Mesenchymal Stem Cell: A Review.

Introduction: The purpose of this study is to investigate the effect of a low-power laser on the proliferation, migration, differentiation of different types of mesenchymal stem cells (MSCs) in different studies. Methods: The relevant articles that were published from 2004 to 2019 were collected from the sources of PubMed, Scopus, and only the articles specifically examining the effect of a lowpower laser on the proliferation, differentiation, and migration of the MSCs were investigated. Results: After reviewing the literature, only 42 articles were found relevant. Generally, most of the studies demonstrated that different laser parameters increased the proliferation, migration, and differentiation of the MSCs, except the results of two studies which were contradictory. In fact, changing the parameters of a low-power laser would affect the results. On the other hand, the source of the stem cells was reported as a key factor. In addition, the combination of lasers with other therapeutic approaches was found to be more effective. Conclusion: The different parameters of lasers has been found to be effective in the proliferation, differentiation, and migration of the MSCs and in general, a low-power laser has a positive effect on the MSCs, helping to improve different disease models.

Potential Mechanisms for Marbling Content Differences in M. Longissimus Dorsi From Wagyu And Brahman Cattle

ObjectivesIntramuscular fat (marbling) affects consumer perceptions of meat quality. Recent studies indicated that intramuscular adipocytes are derived from fibroadipogenic progenitors (FAPs), a type of bipotent mesenchymal progenitor cells in extracellular matrix of muscle fiber. Platelet-derived growth factor receptor α (PDGFRa) is a marker in identifying FAPs. The amount of PDGFRa positive cells refers to the abundance of FAPs, a key factor determining the intramuscular adipogenic efficiency of the animal. In addition, it has been shown that both satellite cells and skeletal muscle fibers regulate the activity of FAPs. In this study, we aimed to identify some underlying mechanisms for the differences in intramuscular fat accumulation between Wagyu, a high marbling breed, and Brahman, a low marbling breed, through a comparison study.Materials and MethodsFive cattle of each breed approaching mature body composition (Wagyu born January to May 2017; Brahman two 2-yr-old and three 3-yr-old, born April 2017 and 2015) were selected for this study. Biopsy samples of M. Longissimus muscle (LM) were taken after local anesthesia between the 12th and 13th ribs. Fresh samples were frozen in isopentane chilled by liquid nitrogen immediately after trimming fat and connective tissue. Other samples were fixed for 4 h in ice-cold 4% paraformaldehyde, soaked overnight in 30% sucrose solution, and then processed in Optimal Cutting Temperature embedding medium. Frozen samples were stored at –80°C before cryosectioning (5 mm) at –25°C for immunochemical staining. Unfixed muscle tissue sections were only used for muscle fiber determinations by rinsing with 1X Tris Buffer Saline (TBS) 3 times, 5 min each, before blocking in 10% goat serum and 1% bovine serum albumin, followed by overnight incubation with primary antibodies at 4°C. Subsequent secondary antibody staining at room temperature for 1 h. TBS/T (0.3% Triton) was used for membrane permeabilization to identify satellite cells, FAPs, and basement membrane. Mounting media containing 4,6-diamidino-2-phenylindole (DAPI) was used for nucleus staining. Primary antibodies against PDGFRα, paired box 7 (PAX7), laminin, and different isoforms of skeletal muscle myosins were used to identify FAPs, satellite cells, basement membrane, and different types of muscle fiber, respectively. All the sections were visualized under a Nikon inflorescence microscope, and images were analyzed by Image J software to identify different muscle fiber types and positive signals of PDGFRα and PAX7. Minimum diameter of each muscle fiber type was the average of 30 randomly selected fibers per section. Data were analyzed with R-studio using ‘t.test ()’ function with the critical value being equal to 0.05.ResultsWagyu muscle demonstrated a greater (P = 0.01) number of FAPs compared with Brahman muscle. A trend toward a higher (P = 0.06) abundance of satellite cells in Wagyu muscle than in Brahman muscles was also identified. No differences were found in muscle fiber diameter or muscle fiber type composition between these two breeds of cattle.ConclusionThe greater number of FAP cells observed in the LM of Wagyu cattle than in Brahman cattle suggests that the higher marbling content of Wagyu meat is at least partially attributed to the more abundant adipogenic progenitor cells, which increases the capacity and efficiency of intramuscular adipogenesis in Wagyu cattle.

Current clinical applications of adipose-derived stem cells in humans and animals

Abstract Adipose derived stem cells are a type of mesenchymal stem cell that, because of their straightforward isolation procedure and ready availability, have been intensively studied in the recent years regarding their possible clinical applications. Additionally, ADSCs have the ability to differentiate into tri-germ lineages, as well as exhibit paracrine activity. Their capacity to differentiate into many different cell lineages such as osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic cells, and hepatocytes, has granted them a significant place in consideration for tissue engineering and for their application in regenerative medicine. Moreover, their endocrine activity has a great impact upon therapies as it grants immunosuppressive properties and low immunogenicity. Running title: Clinical applications of ADSCs

Treatment with Stem Cells from Human Exfoliated Deciduous Teeth and Their Derived Conditioned Medium Improves Retinal Visual Function and Delays the Degeneration of Photoreceptors.

Retinitis pigmentosa (RP) is a hereditary disease characterized by degeneration and the loss of photoreceptors. Stem cell-based therapy has emerged as a promising strategy for treating RP. Stem cells from exfoliated deciduous teeth (SHEDs), a type of mesenchymal stem cell from human exfoliated deciduous teeth, have the potential to differentiate into photoreceptor-like cells under specific induction in vitro. It has been confirmed that through paracrine secreta, SHEDs exert neurotrophic, angiogenic, immunoregulatory, and antiapoptotic functions in injured tissues. This study was designed to determine whether retinal-differentiated SHEDs and the conditioned medium derived from SHEDs (SHEDs-CM) have therapeutic effects in a mouse model of RP. The results showed that both SHEDs and SHEDs-CM improved electroretinogram responses, ameliorated photoreceptor degeneration, and maintained the structure of the outer segments of photoreceptors. The therapeutic effects were related to antiapoptotic activity of SHEDs and SHEDs-CM. Thus, SHEDs may be a promising stem cell source for treating retinal degeneration.

Isolation and Characterization of Mesenchymal Stem Cells from Human Liposuction Aspirate

Background: Adipose derived stem cells (ADSCs) are a new type of mesenchymal stem cells (MSC) resemble bone marrow stem cells in their biologic activities. DScs is multipotent stem cells can differentiate into multiple cell types including osteoblasts, chondrocytes, adipocytes, myocytes, vascular endothelial cells, and neurons. Stromal vascular fraction (SVF) corresponds to ADSC and describes the cells obtained immediately from adipose tissues after the digestion of collagenase. Adipose tissue is a promising source of adult mesenchymal stem cells for therapeutic applications because it is available in large amounts (100 ml up to 1 liter) through liposuction and with minimal morbidity. Aim: To isolate and characterize the adipose-derived stem cells from human liposuction aspirate. Material and Methods: liposuction aspirate was used for the extraction of the stromal vascular fragment (SVF). SVF was used for culturing and isolation of hADSCs.ADSCs were cultured in tissue-culture flasks using collagenase digestion method. Flow cytometric analysis was used to identify ADSCs markers using fluorescence-activated cell sorting (FACS)against CD44, CD90, and CD45. Results: Human ADSCs were isolated successfully using collagenase digestion method. After 4 days the cells appeared to be spindle in shape and formed symmetric colonies. After 7 days, the cell sheet formed 60% confluence and completed to 90% confluence after 10 days incubation. FACS analysis of ADSCs surface markers revealed expression of CD44, and CD90 markers in most of the adherent cells. In contrast, most adherent cells were negative for CD45. Conclusion: Human ADSCs were isolated successfully from human lipoaspirate. Keywords: ADSCs; MSC; SVF

Knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes in the Mdr2−/− mouse model of primary sclerosing cholangitis (PSC)

BackgroundCholangiocytes are the target cells of cholangiopathies including primary sclerosing cholangitis (PSC). Vimentin is an intermediate filament protein that has been found in various types of mesenchymal cells. The aim of this study is to evaluate the role of vimentin in the progression of biliary damage/liver fibrosis and whether there is a mesenchymal phenotype of cholangiocytes in the Mdr2−/− model of PSC.MethodsIn vivo studies were performed in 12 wk. Mdr2−/− male mice with or without vimentin Vivo-Morpholino treatment and their corresponding control groups. Liver specimens from human PSC patients, human intrahepatic biliary epithelial cells (HIBEpiC) and human hepatic stellate cell lines (HHSteCs) were used to measure changes in epithelial-to-mesenchymal transition (EMT).FindingsThere was increased mesenchymal phenotype of cholangiocytes in Mdr2−/− mice, which was reduced by treatment of vimentin Vivo-Morpholino. Concomitant with reduced vimentin expression, there was decreased liver damage, ductular reaction, biliary senescence, liver fibrosis and TGF-β1 secretion in Mdr2−/− mice treated with vimentin Vivo-Morpholino. Human PSC patients and derived cell lines had increased expression of vimentin and other mesenchymal markers compared to healthy controls and HIBEpiC, respectively. In vitro silencing of vimentin in HIBEpiC suppressed TGF-β1-induced EMT and fibrotic reaction. HHSteCs had decreased fibrotic reaction and increased cellular senescence after stimulation with cholangiocyte supernatant with reduced vimentin levels.InterpretationOur study demonstrated that knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes, which leads to decreased biliary senescence and liver fibrosis. Inhibition of vimentin may be a key therapeutic target in the treatment of cholangiopathies including PSC.FundNational Institutes of Health (NIH) awards, VA Merit awards.

Comparison of Rapidly Proliferating, Multipotent Aortic Valve-Derived Stromal Cells and Valve Interstitial Cells in the Human Aortic Valve.

Aortic valve calcification is a common clinical disease, caused by valve interstitial cells (VICs), which initiate the thickening and then calcification of valve leaflets. Classical valve-derived cells can be seen in different cell populations according to their different morphologies, but it is not clear whether different types of mesenchymal cells exist. In this study, culture conditions for mesenchymal stromal cells were used to selectively isolate valve-derived stromal cells (VDSCs). After subculturing, the morphology, proliferation, multidifferentiation, immunophenotype, and gene expression profiling in isolated VDSCs were compared with those in conventional cultured VICs. VDSCs isolated from human aortic valves were uniform spindle-shaped fibroblasts, had mutilineage differentiation abilities, and proliferated faster than VICs. Classic mesenchymal markers including cluster of differentiation 90 (CD90), CD44, and CD29 were positively expressed. In addition, the stem cell markers CD163, CD133, and CD106 were all expressed in VDSCs. RNA-sequencing identified 1595 differentially expressed genes between VDSCs and VICs of which 301 were upregulated and 1294 were downregulated. Valvular extracellular matrix genes of VDSCs such as collagen type 1, alpha 1 (COL1A1), COL1A2, and fibronectin 1 were abundantly expressed. In addition, runt-related transcription factor 2 and Ki-67 proteins were also markedly upregulated in VDSCs, whereas there was less expression of the focal adhesion genes integrin alpha and laminin alpha in VDSCs compared to VICs. In conclusion, novel rapidly proliferating VDSCs with fibroblast morphology, which were found to express mesenchymal and osteogenic markers, may contribute to aortic valve calcification.

Two-dimensional plasmonic biosensing platform: Cellular activity detection under laser stimulation

Combining biosensors with nanoscience provides great advantages such as being label-free and real time, highly sensitive, and small in size, as well as providing a low limit of detection and integration to other systems. That is why plasmonics finds various applications in drug detection, food safety, agriculture, photothermal therapy, etc. In this paper, we have fabricated a two-dimensional plasmonic grating biosensor using a soft lithography technique, which has eliminated some disadvantages of conventional plasmonic structures like expensive fabrication cost, inflexibility, and lack of mass production. On the other hand, we benefited from infrared neural stimulation for regulating membrane depolarization, which was based on photothermal mechanism and provided a contact-free and high spatial/temporal resolution. Eventually, the membrane depolarization of two different cell types of Hep G2 and mesenchymal stem cells cultured on two-dimensional plasmonic structure has been investigated under infrared neural stimulation. After preparing the soft plasmonic crystal, its reflection spectra and respective ellipsometry parameters were analyzed before and after cell culture with/without stimulation (near-infrared immune region ∼1450 nm). By comparing the obtained ellipsometry results for HEP G2 and mesenchymal stem cells, it is observed that the behavior of two cell types with respect to IR stimulation was the same as well as providing us the possibility of distinguishing the level of membrane depolarization under various stimulating frequencies. The strength of this integrated system for membrane depolarization detection has been shown experimentally, which can open new avenues toward neuroplasmonic application in the future.

Induced pluripotent stem cell-derived mesenchymal stem cells prolong hind limb survival in a rat vascularized composite allotransplantation model.

The reduction of systemic immunosuppressive agents is essential for the expansion of vascularized composite allotransplantation (VCA) in a clinical setting. The purpose of this study is to compare human-induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) with four other types of mesenchymal stem cells (human bone marrow-derived MSCs [BMMSCs], human adipose-derived MSCs [ADMSCs], rat BMMSCs, and rat ADMSCs) in vitro, and to investigate the in vivo immunomodulatory effect of iMSCs in a rat VCA model. One Brown Norway (BN) rat, 2 Lewis (LEW) rats, and 1 Wistar rat were used in the mixed lymphocyte reaction (MLR), and 9 BN rats and 3 LEW rats (for donors), and 24 LEW rats (for recipients) were used in the VCA model. The abovementioned five types of MSCs were imaged to examine their morphology and were also tested for suppressor function using a MLR. The 24 recipient LEW rats were divided randomly into four groups, and subjected to orthotopic hind limb transplantation. The three control groups were the Iso group, in which transplantation was performed on from three to six LEW rats without immunosuppressive treatment (n = 6); the FK group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus alone (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) (n = 6); and the UT group, in which transplantation was performed from BN rats to LEW rats without any immunosuppressive treatment (n = 6). The experimental group was the iMSC group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) and injected with iMSCs (2 × 106 cells, day 7, intravenously) (n = 6). Hind limb survival was assessed by daily inspection of gross appearance until 50 days postoperatively. Histology of the skin and muscle biopsy were investigated on day 14 postoperatively. A time series of the plasma cytokine level (before transplantation, and at 10, 14, and 17 days after transplantation) was also analyzed. The size of adherent and trypsinized iMSCs was 67.5 ± 8.7 and 9.5 ± 1.1 μm, respectively, which was the smallest among the five types of MSCs (p < .01). The absorbance in MLR was significantly smaller with rat ADMSCs (p = .0001), human iMSCs (p = .0006), rat BMMSCs (p = .0014), human ADMSCs (p = .0039), and human BMMSCs (p = .1191) compared to without MSCs. In vivo, iMSC treatment prolonged hind limb survival up to 12.7 days in macroscopic appearance, which is significantly longer than that of the FK group (p < .01). Histology of the skin and muscle biopsy revealed that mononuclear cell infiltration was significantly reduced by iMSC injection (p < .01). iMSC treatment also affected proinflammatory cytokines (interferon-gamma (IFNγ) and tumor necrosis factor α (TNFα)) and the anti-inflammatory cytokine (interleukin-10 (IL-10)) of the recipient plasma. The IFNγ levels at Δ14 and the TNFα levels at Δ14 and Δ17 of the iMSC group were significantly lower than those of the FK group (p = .0226, .0004, and .004, respectively). The IL-10 levels at Δ10 and Δ14 of the iMSC group were significantly higher than those of the FK group (p = .0013 and .0374, respectively). iMSCs induce T cell hyporesponsiveness to prolong hind limb survival in a rat VCA model. This immunomodulatory property against acute rejection could provide one of the promising strategies capable of enabling the toxicities of immunosuppressants to be avoided in clinical settings.

SURG-03. SPATIAL COORDINATES FROM GAMMA KNIFE RADIOSURGERY REVEAL PRIMARY CANCERS HAVE REGIONAL CNS TOPOGRAPHICAL DISTRIBUTION FOR BRAIN METASTASES

Brain metastases arise in the central nervous system (CNS) following spread of circulating mesenchymal-type cells from primary tumors. While accumulating evidence underlines the importance of the neural niche in the establishment and progression of metastases, there still remains ambiguity over CNS anatomical spatial distribution from primary cancers. We evaluated 973 patients with brain metastases (breast, colorectal, lung, melanoma, renal) totaling 2,106 lesions treated from 1994–2015 with gamma knife radiosurgery at the University of Southern California Keck Medical Center for topographical distribution analysis. MRI images of the brain were taken and used in conjunction with the frame to precisely localize tumors and measure their size. Each tumor was given an x, y, and z-coordinate derived from the head frame that corresponded to its volumetric center within a 3-dimensional Cartesian field. Topographical analyses were conducted using logistic and multinomial spatial generalized additive models (GAM). For each cancer origin type we compared the observed brain metastases to set of randomly generated spatial observations to determine whether there were statistically significant localization patterns. Spatial pattern results show: 1) melanoma has highest probability to metastasize to the right frontal (74.5%, 95% confidence interval [Cl] = 63.6%- 85.4%) and to occipital lobe (72.4%, 95% Cl = 65.8%-78.9%), 2) while breast cancers have highest proclivity to metastasize to left cerebellar hemisphere (25%, 95% Cl=16.0%-34.1%) and brainstem (16.6%, 95% Cl= 10.8%-22.4%), 3) with lung tumors metastasizing to the left (23.7%, 95% Cl= 16.0–31.3%) and right parietal (24.7%, 95% Cl=16.7–32.8%), left temporal lobe (25.2%, 95% Cl=21.4%-29.1%). Colon and renal metastases show weak spatial patterns across the CNS. We conclude there is evidence of non-uniform spatial distribution of metastasis in the brain. These tumor-specific CNS topography patterns may underlie the ability of cancer cells to adapt to the regional neural microenvironments in order to facilitate colonization and establishment of metastasis.

Heterocellular gene signatures reveal luminal-A breast cancer heterogeneity and differential therapeutic responses

Breast cancer is a highly heterogeneous disease. Although differences between intrinsic breast cancer subtypes have been well studied, heterogeneity within each subtype, especially luminal-A cancers, requires further interrogation to personalize disease management. Here, we applied well-characterized and cancer-associated heterocellular signatures representing stem, mesenchymal, stromal, immune, and epithelial cell types to breast cancer. This analysis stratified the luminal-A breast cancer samples into five subtypes with a majority of them enriched for a subtype (stem-like) that has increased stem and stromal cell gene signatures, representing potential luminal progenitor origin. The enrichment of immune checkpoint genes and other immune cell types in two (including stem-like) of the five heterocellular subtypes of luminal-A tumors suggest their potential response to immunotherapy. These immune-enriched subtypes of luminal-A tumors (containing only estrogen receptor positive samples) showed good or intermediate prognosis along with the two other differentiated subtypes as assessed using recurrence-free and distant metastasis-free patient survival outcomes. On the other hand, a partially differentiated subtype of luminal-A breast cancer with transit-amplifying colon-crypt characteristics showed poor prognosis. Furthermore, published luminal-A subtypes associated with specific somatic copy number alterations and mutations shared similar cellular and mutational characteristics to colorectal cancer subtypes where the heterocellular signatures were derived. These heterocellular subtypes reveal transcriptome and cell-type based heterogeneity of luminal-A and other breast cancer subtypes that may be useful for additional understanding of the cancer type and potential patient stratification and personalized medicine.

Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-Like Cells for Allogeneic Cell Therapies.

Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, we evaluated their inductive and modulatory capacity regarding immune responses and underlying key mechanisms in vitro. For this, cryopreserved hAACs were either cultured in the presence of interferon-gamma (IFNγ) or left unstimulated. The expression of characteristic mesenchymal stromal cell markers (CD29, CD44, CD73, CD105, CD166) was revealed by flow cytometry that also highlighted a predominant negativity for CD90. A low immunogeneic phenotype in an inflammatory milieu was shown by lacking expression of co-stimulatory molecules and upregulation of the inhibitory ligands PD-L1 and PD-L2, despite de novo expression of HLA-DR. Co-cultures of hAACs with allogeneic peripheral blood mononuclear cells, proved their low immunogeneic state by absence of induced T cell proliferation and activation. Additionally, elevated levels of IL-1β, IL-33, and IL-10 were detectable in those cell culture supernatants. Furthermore, the immunomodulatory potential of hAACs was assessed in co-cultures with αCD3/αCD28-activated peripheral blood mononuclear cells. Here, a strong inhibition of T cell proliferation and reduction of pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-17A, IL-2) were observable after pre-stimulation of hAACs with IFNγ. Transwell experiments confirmed that mostly soluble factors are responsible for these suppressive effects. We were able to identify indolamin-2,3-dioxygenase (IDO) as a potential key player through a genome-wide gene expression analysis and could demonstrate its involvement in the observed immunological responses. While the application of blocking antibodies against both PD-1 ligands did not affect the immunomodulation by hAACs, 1-methyl-L-tryptophan as specific inhibitor of IDO was able to restore proliferation and to lower apoptosis of T cells. In conclusion, hAACs represent a cardiac-derived mesenchymal stromal-like cell type with a high potential for the application in an allogeneic setting, since they do not trigger T cell responses and even increase their immunomodulatory potential in inflammatory environments.