Human primitive mesenchymal stem cell-derived retinal progenitor cells improved neuroprotection, neurogenesis, and vision in rd12 mouse model of retinitis pigmentosa.

BACKGROUND: Currently, there is no treatment for retinal degenerative diseases (RDD) such as retinitis pigmentosa (RP). Stem cell-based therapies could provide promising opportunities to repair the damaged retina and restore vision. Thus far, primarily adult mesenchymal stem cells (MSCs) have been investigated in preclinical and clinical studies, and the results have not been convincing. We applied a new approach in which primitive (p) MSC-derived retinal progenitor cells (RPCs) were examined to treat retinal degeneration in an rd12 mouse model of RP. METHODS: Well-characterized pMSCs and RPCs labeled with PKH26 were intravitreally injected into rd12 mice. The vision and retinal function of transplanted animals were analyzed using electroretinography. Animals were killed 4 and 8 weeks after cell transplantation for histological, immunological, molecular, and transcriptomic analyses of the retina. RESULTS: Transplanted RPCs significantly improved vision and retinal thickness as well as function in rd12 mice. pMSCs and RPCs homed to distinct retinal layers. pMSCs homed to the retinal pigment epithelium, and RPCs migrated to the neural layers of the retina, where they improved the thickness of the respective layers and expressed cell-specific markers. RPCs induced anti-inflammatory and neuroprotective responses as well as upregulated the expression of genes involved in neurogenesis. The transcriptomic analysis showed that RPCs promoted neurogenesis and functional recovery of the retina through inhibition of BMP and activation of JAK/STAT and MAPK signaling pathways. CONCLUSIONS: Our study demonstrated that RPCs countered inflammation, provided retinal protection, and promoted neurogenesis resulting in improved retinal structure and physiological function in rd12 mice.

Neural stem cells derived from primitive mesenchymal stem cells reversed disease symptoms and promoted neurogenesis in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). MS affects millions of people and causes a great economic and societal burden. There is no cure for MS. We used a novel approach to investigate the therapeutic potential of neural stem cells (NSCs) derived from human primitive mesenchymal stem cells (MSCs) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. METHODS: MSCs were differentiated into NSCs, labeled with PKH26, and injected into the tail vein of EAE mice. Neurobehavioral changes in the mice assessed the effect of transplanted cells on the disease process. The animals were sacrificed two weeks following cell transplantation to collect blood, lymphatic, and CNS tissues for analysis. Transplanted cells were tracked in various tissues by flow cytometry. Immune infiltrates were determined and characterized by H&E and immunohistochemical staining, respectively. Levels of immune regulatory cells, Treg and Th17, were analyzed by flow cytometry. Myelination was determined by Luxol fast blue staining and immunostaining. In vivo fate of transplanted cells and expression of inflammation, astrogliosis, myelination, neural, neuroprotection, and neurogenesis markers were investigated by using immunohistochemical and qRT-PCR analysis. RESULTS: MSC-derived NSCs expressed specific neural markers, NESTIN, TUJ1, VIMENTIN, and PAX6. NSCs improved EAE symptoms more than MSCs when transplanted in EAE mice. Post-transplantation analyses also showed homing of MSCs and NSCs into the CNS with concomitant induction of an anti-inflammatory response, resulting in reducing immune infiltrates. NSCs also modulated Treg and Th17 cell levels in EAE mice comparable to healthy controls. Luxol fast blue staining showed significant improvement in myelination in treated mice. Further analysis showed that NSCs upregulated genes involved in myelination and neuroprotection but downregulated inflammatory and astrogliosis genes more significantly than MSCs. Importantly, NSCs differentiated into neural derivatives and promoted neurogenesis, possibly by modulating BDNF and FGF signaling pathways. CONCLUSIONS: NSC transplantation reversed the disease process by inducing an anti-inflammatory response and promoting myelination, neuroprotection, and neurogenesis in EAE disease animals. These promising results provide a basis for clinical studies to treat MS using NSCs derived from primitive MSCs.

Transcriptomic Analysis of Naïve Human Embryonic Stem Cells Cultured in Three-Dimensional PEG Scaffolds.

Naïve human embryonic stem cells (ESCs) are characterized by improved viability, proliferation, and differentiation capacity in comparison to traditionally derived primed human ESCs. However, currently used two-dimensional (2-D) cell culture techniques fail to mimic the three-dimensional (3-D) in vivo microenvironment, altering morphological and molecular characteristics of ESCs. Here, we describe the use of 3-D self-assembling scaffolds that support growth and maintenance of the naïve state characteristics of ESC line, Elf1. Scaffolds were formed via a Michael addition reaction upon the combination of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups. 3-D scaffold environment maintained the naïve state and supported the long-term growth of ESCs. RNA-sequencing demonstrated significant changes in gene expression profiles between 2-D and 3-D grown cells. Gene ontology analysis revealed upregulation of biological processes involved in the regulation of transcription and translation, extracellular matrix organization, and chromatin remodeling in 3-D grown cells. 3-D culture conditions also induced upregulation of genes associated with Wnt and focal adhesion signaling, while p53 signaling pathway associated genes were downregulated. Our findings, for the first time, provide insight into the possible mechanisms of self-renewal of naïve ESCs stimulated by the transduction of mechanical signals from the 3-D microenvironment.

Mesenchymal stem cells: Cell therapy and regeneration potential.

Rapid advances in the isolation of multipotent progenitor cells, routinely called mesenchymal stromal/stem cells (MSCs), from various human tissues and organs have provided impetus to the field of cell therapy and regenerative medicine. The most widely studied sources of MSCs include bone marrow, adipose, muscle, peripheral blood, umbilical cord, placenta, fetal tissue, and amniotic fluid. According to the standard definition of MSCs, these clonal cells adhere to plastic, express cluster of differentiation (CD) markers such as CD73, CD90, and CD105 markers, and can differentiate into adipogenic, chondrogenic, and osteogenic lineages in vitro. However, isolated MSCs have been reported to vary in their potency and self-renewal potential. As a result, the MSCs used for clinical applications often lead to variable or even conflicting results. The lack of uniform characterization methods both in vitro and in vivo also contributes to this confusion. Therefore, the name "MSCs" itself has been increasingly questioned lately. As the use of MSCs is expanding rapidly, there is an increasing need to understand the potential sources and specific potencies of MSCs. This review discusses and compares the characteristics of MSCs and suggests that the variations in their distinctive features are dependent on the source and method of isolation as well as epigenetic changes during maintenance and growth. We also discuss the potential opportunities and challenges of MSC research with the hope to stimulate their use for therapeutic and regenerative medicine.

Self-Assembling Scaffolds Supported Long-Term Growth of Human Primed Embryonic Stem Cells and Upregulated Core and Naïve Pluripotent Markers.

The maintenance and expansion of human embryonic stem cells (ESCs) in two-dimensional (2-D) culture is technically challenging, requiring routine manipulation and passaging. We developed three-dimensional (3-D) scaffolds to mimic the in vivo microenvironment for stem cell proliferation. The scaffolds were made of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups, which self-assembled via a Michael addition reaction. When primed ESCs (H9 cells) were mixed with PEG polymers, they were encapsulated and grew for an extended period, while maintaining their viability, self-renewal, and differentiation potential both in vitro and in vivo. Three-dimensional (3-D) self-assembling scaffold-grown cells displayed an upregulation of core pluripotency genes, OCT4, NANOG, and SOX2. In addition, the expression of primed markers decreased, while the expression of naïve markers substantially increased. Interestingly, the expression of mechanosensitive genes, YAP and TAZ, was also upregulated. YAP inhibition by Verteporfin abrogated the increased expression of YAP/TAZ as well as core and naïve pluripotent markers. Evidently, the 3-D culture conditions induced the upregulation of makers associated with a naïve state of pluripotency in the primed cells. Overall, our 3-D culture system supported the expansion of a homogenous population of ESCs and should be helpful in advancing their use for cell therapy and regenerative medicine.

Potential of Human Nucleus Pulposus-Like Cells Derived From Umbilical Cord to Treat Degenerative Disc Disease.

BACKGROUND: Degenerative disc disease (DDD) is a common spinal disorder that manifests with neck and lower back pain caused by the degeneration of intervertebral discs (IVDs). Currently, there is no treatment to cure this debilitating ailment. OBJECTIVE: To investigate the potential of nucleus pulposus (NP)-like cells (NPCs) derived from human umbilical cord mesenchymal stem cells (MSCs) to restore degenerated IVDs using a rabbit DDD model. METHODS: NPCs differentiated from MSCs were characterized using quantitative real-time reverse transcription polymerase chain reaction and immunocytochemical analysis. MSCs and NPCs were labeled with fluorescent dye, PKH26, and transplanted into degenerated IVDs of a rabbit model of DDD (n = 9 each). Magnetic resonance imaging of the IVDs was performed before and after IVD degeneration, and following cell transplantation. IVDs were extracted 8 wk post-transplantation and analyzed by various biochemical, immunohistological, and molecular techniques. RESULTS: NPC derivatives of MSCs expressed known NP-specific genes, SOX9, ACAN, COL2, FOXF1, and KRT19. Transplanted cells survived, dispersed, and integrated into the degenerated IVDs. IVDs augmented with NPCs showed significant improvement in the histology, cellularity, sulfated glycosaminoglycan and water contents of the NP. In addition, expression of human genes, SOX9, ACAN, COL2, FOXF1, KRT19, PAX6, CA12, and COMP, as well as proteins, SOX9, ACAN, COL2, and FOXF1, suggest NP biosynthesis due to transplantation of NPCs. Based on these results, a molecular mechanism for NP regeneration was proposed. CONCLUSION: The findings of this study demonstrating feasibility and efficacy of NPCs to regenerate NP should spur interest for clinical studies to treat DDD using cell therapy.

Modulating affective experience and emotional intelligence with loving kindness meditation and transcranial direct current stimulation: A pilot study.

Positive emotional perceptions and healthy emotional intelligence (EI) are important for social functioning. In this study, we investigated whether loving kindness meditation (LKM) combined with anodal transcranial direct current stimulation (tDCS) would facilitate improvements in EI and changes in affective experience of visual stimuli. LKM has been shown to increase positive emotional experiences and we hypothesized that tDCS could enhance these effects. Eighty-seven undergraduates were randomly assigned to 30 minutes of LKM or a relaxation control recording with anodal tDCS applied to the left dorsolateral prefrontal cortex (left dlPFC) or right temporoparietal junction (right TPJ) at 0.1 or 2.0 milliamps. The primary outcomes were self-reported affect ratings of images from the International Affective Picture System and EI as measured by the Mayer, Salovey and Caruso Emotional Intelligence Test. Results indicated no effects of training on EI, and no main effects of LKM, electrode placement, or tDCS current strength on affect ratings. There was a significant interaction of electrode placement by meditation condition (p = 0.001), such that those assigned to LKM and right TPJ tDCS, regardless of current strength, rated neutral and positive images more positively after training. Results suggest that LKM may enhance positive affective experience.

Toxicity of JQ1 in neuronal derivatives of human umbilical cord mesenchymal stem cells.

Bromodomain and extra-terminal domain (BET) proteins regulate the transcription of many genes including c-MYC, a proto-oncogene, which is upregulated in many types of cancers. The thienodiazepine class of BET inhibitors, such as JQ1, inhibits growth of cancer cells and triggers apoptosis. However, the effects of BET inhibitors on normal cells and mesenchymal stem cells (MSCs), which are important in routine maintenance or regeneration of damaged cells and tissues, are poorly investigated. Previously, we have shown that JQ1 causes human umbilical cord MSCs to undergo cell cycle arrest and neural differentiation. In this study, we determined that JQ1 is more deleterious to neuronal derivatives (NDs) than adipogenic, chondrogenic or osteogenic derivatives of MSCs. NDs treated with JQ1 showed a significant decrease in cell proliferation, viability, and neuronal markers. JQ1 caused cell death through the intrinsic apoptotic pathway in NDs as determined by activation of Caspase 9 and increased expression of Cytochrome C. A comparative analysis showed differential action of JQ1 on MSCs and NDs. The results showed selective neuronal toxicity of JQ1 in NDs but not in the undifferentiated MSCs. These findings suggest a more careful examination of the selection and use of BET inhibitors as therapeutic agents, as they may cause unwanted damage to non-target cells and tissues.

Cytotoxicity of radiocontrast dyes in human umbilical cord mesenchymal stem cells.

Radiocontrast dyes are used for a wide range of diagnostic procedures for enhancing the image of anatomical structures, pain targets, and vascular uptake. While some of these dyes show toxicity to primary cells, their effect on stem cells, particularly mesenchymal stem cells (MSCs), is unknown. This study investigates the cytotoxic effects of two clinically used radiocontrast dyes, iohexol and iopamidol, on bone marrow and human umbilical cord MSCs. Exposure to these dyes significantly affected morphology of MSCs from both sources, as treated cells appeared transparent and no longer fibroblastoid. Cell viability decreased as determined by trypan blue and Annexin-V/PI staining, in a dose dependent manner with simultaneous loss of CD90 and CD105 concurrent with spontaneous differentiation in MSCs treated with iohexol and iopamidol. In addition, significantly higher cell death was observed in MSCs exposed to iopamidol than iohexol. At a concentration of 1:1, iohexol and iopamidol induced apoptosis in 19% and 92% (<.01) of MSCs, respectively. Global transcriptome analysis of treated MSCs revealed 139 and 384 differentially expressed genes in iohexol vs control and iopamidol vs control at p ≤ .01 and 1.5-fold, respectively. This suggested that iopamidol had more significant effect on the transcription of MSCs. Based on these results a molecular mechanism of radiocontast dye induced cell death via intrinsic apoptosis pathway mediated by p53 was proposed. Since iopamidol was significantly more toxic than iohexol in human MSCs, a more careful examination of safety of radiocontrast dyes for clinical use is warranted.

The effects of black cohosh on the regulation of estrogen receptor (ERα) and progesterone receptor (PR) in breast cancer cells.

The North American plant Cimicifuga racemosa, also known as black cohosh (BC), is a herb that recently has gained attention for its hormonal effects. As the usage of hormone replacement therapy is declining due to its adverse effects in women with cancer, many are turning to herbal remedies like BC to treat menopausal symptoms. It is crucial to determine whether the effects of BC involve estrogen receptor-alpha (ERα). Previous studies from our laboratory have shown ERα to be a possible molecular target for BC. In this study, we examined the effects of BC (8% triterpene glycosides) alone and in combination with hormones and antihormones on the cellular viability, expression of ERα and progesterone receptor (PR)-A/B, and cytolocalization of ERα in ER (+) and PR-A/B (+) T-47D breast cancer cells. Cells were cultured and proteins were extracted and quantified. Western blot analysis revealed alterations in the expression of ERα and PR after treatment with BC (5-100 µM). BC induced a concentration-dependent decrease in ERα and PR protein levels when compared to the control. Image cytometric analysis with propidium iodide staining was used to enumerate changes in T-47D cell number and viability. A decrease in T-47D cell viability was observed upon treatment with 5-100 µM BC. The ideal concentration of BC (100 µM) was used in combination with hormones and antihormones in an effort to further understand the possible similarities between this compound and other known effectors of ERα and PR. After a 24-hour concomitant treatment with and/or in combination of BC, estradiol, ICI 182, 780, and Tamoxifen, downregulation of ERα and PR protein levels was observed. Delineating the role of BC in the regulation of ERα, PR, as well as its mechanisms of action, may be important in understanding the influence of BC on hormone receptors in breast cancer.

Human umbilical cord derivatives regenerate intervertebral disc.

Intervertebral disc (IVD) degeneration is characterized by the loss of nucleus pulposus (NP), which is a common cause for lower back pain. Although, currently, there is no cure for the degenerative disc disease, stem cell therapy is increasingly being considered for its treatment. In this study, we investigated the feasibility and efficacy of human umbilical cord mesenchymal stem cells (MSCs) and chondroprogenitor cells (CPCs) derived from those cells to regenerate damaged IVD in a rabbit model. Transplanted cells survived, engrafted and dispersed into NP in situ. Significant improvement in the histology, cellularity, extracellular matrix proteins, and water and glycosaminoglycan contents in IVD recipients of CPCs was observed compared to MSCs. In addition, IVDs receiving CPCs exhibited higher expression of NP-specific human markers, SOX9, aggrecan, collagen 2, FOXF1 and KRT19. The novelty of the study is that in vitro differentiated CPCs derived from umbilical cord MSCs, demonstrated far greater capacity to regenerate damaged IVDs, which provides basis and impetus for stem cell based clinical studies to treat degenerative disc disease. Copyright © 2016 John Wiley & Sons, Ltd.

Advances and challenges in stem cell culture.

Stem cells (SCs) hold great promise for cell therapy, tissue engineering, and regenerative medicine as well as pharmaceutical and biotechnological applications. They have the capacity to self-renew and the ability to differentiate into specialized cell types depending upon their source of isolation. However, use of SCs for clinical applications requires a high quality and quantity of cells. This necessitates large-scale expansion of SCs followed by efficient and homogeneous differentiation into functional derivatives. Traditional methods for maintenance and expansion of cells rely on two-dimensional (2-D) culturing techniques using plastic culture plates and xenogenic media. These methods provide limited expansion and cells tend to lose clonal and differentiation capacity upon long-term passaging. Recently, new approaches for the expansion of SCs have emphasized three-dimensional (3-D) cell growth to mimic the in vivo environment. This review provides a comprehensive compendium of recent advancements in culturing SCs using 2-D and 3-D techniques involving spheroids, biomaterials, and bioreactors. In addition, potential challenges to achieve billion-fold expansion of cells are discussed.

Mechanism of arsenite toxicity in embryonic stem cells.

Environmental arsenite exposure has been linked to cancer as well as other diseases, presenting an important and serious public health problem. Toxicity of inorganic arsenite (iAs) has been investigated using animal models and cell culture, yet its developmental effects are poorly understood. This study investigated the molecular mechanism of iAs toxicity to ascertain insight into development and differentiation processes using mouse embryonic stem cells (ESCs). The results showed that iAs exposure affected morphology and integrity of ESC colonies as well as inhibited cell growth in a concentration-dependent manner, excluding concentrations <1 µM iAs which stimulated ESC growth. ESCs self-renewal and pluripotency was also affected as evident from the downregulation of transcription circuitry, Oct4, Nanog, Sox2 and Klf4 resulting in non-specific differentiation. ESCs exposed to iAs randomly differentiated into three germ layers, mesoderm, endoderm and ectoderm, as judged by transcriptional expression of Brachyury, Gata4 and FGF2, as well as translational expression of BRACHYURY, GATA4 and TUJ1 respectively. The differentiated cells represented osteogenic, chondrogenic, myogenic and neurogenic lineages as evident from upregulation of Col1, Sox9, Col2, Myog, Notch, Nes and Nef. Although iAs caused slight apoptosis with a concomitant increase in ROS levels, the exposed ESCs had significant Bcl2 expression, which could be involved in the protection against apoptosis. Further analysis revealed upregulation of Jun and P38 in ESCs with an increase in iAs concentration. These observations indicated that iAs stress caused random differentiation of ESCs via JNK/P38 pathways. These findings suggest that iAs exposure may cause teratogenicity during early fetal development. Copyright © 2017 John Wiley & Sons, Ltd.

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton's jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Compression Induced Chondrogenic Differentiation of Embryonic Stem Cells in Three-Dimensional Polydimethylsiloxane Scaffolds.

Embryonic stem cells (ESCs) are an ideal source for chondrogenic progenitors for the repair of damaged cartilage tissue. It is currently difficult to induce uniform and scalable ESC differentiation in vitro, a process required for stem cell therapy. This is partly because stem cell fate is determined by complex interactions with the native microenvironment and mechanical properties of the extracellular matrix. Mechanical signaling is considered to be one of the major factors regulating the proliferation and differentiation of chondrogenic cells both in vitro and in vivo. We used biocompatible and elastic polydimethylsiloxane (PDMS) scaffolds, capable of transducing mechanical signals, including compressive stress in vitro. ESCs seeded into the PDMS scaffolds and subjected to mechanical loading resulted in induction of differentiation. Differentiated ESC derivatives in three-dimensional (3-D) PDMS scaffolds exhibited elongated single cell rather than round clonal ESC morphology. They expressed chondrogenic marker, Col2, with concomitant reduction in the expression of pluripotent marker, Oct4. Immunocytochemical analysis also showed that the expression of COL2 protein was significantly higher in ESCs in 3-D scaffolds subjected to compressive stress. Further analysis showed that compressive stress also resulted in expression of early chondrogenic makers, Sox9 and Acan, but not hypertrophic chondrogenic markers, Runx2, Col10, and Mmp13. Compressive stress induced differentiation caused a reduction in the expression of ß-Catenin and an increase in the expression of genes, Rhoa, Yap, and Taz, which are known to be affected by mechanosignaling. The chondroinductive role of RhoA was confirmed by its downregulation with simultaneous decrease in the transcriptional and translational expression of early chondrogenic markers, SOX9, COL2, and ACAN, when ESCs in PDMS scaffolds were subjected to compressive stress and treated with RhoA inhibitor, CCG-1432. Based on these observations, a model for compression induced chondrogenic differentiation of ESCs in 3-D scaffolds was proposed.

Isolation and comparative analysis of potential stem/progenitor cells from different regions of human umbilical cord.

Human umbilical cord (hUC) blood and tissue are non-invasive sources of potential stem/progenitor cells with similar cell surface properties as bone marrow stromal cells (BMSCs). While they are limited in cord blood, they may be more abundant in hUC. However, the hUC is an anatomically complex organ and the potential of cells in various sites of the hUC has not been fully explored. We dissected the hUC into its discrete sites and isolated hUC cells from the cord placenta junction (CPJ), cord tissue (CT), and Wharton's jelly (WJ). Isolated cells displayed fibroblastoid morphology, and expressed CD29, CD44, CD73, CD90, and CD105, and showed evidence of differentiation into multiple lineages in vitro. They also expressed low levels of pluripotency genes, OCT4, NANOG, SOX2 and KLF4. Passaging markedly affected cell proliferation with concomitant decreases in the expression of pluripotency and other markers, and an increase in chondrogenic markers. Microarray analysis further revealed the differences in the gene expression of CPJ-, CT- and WJ-hUC cells. Five coding and five lncRNA genes were differentially expressed in low vs. high passage hUC cells. Only MAEL was expressed at high levels in both low and high passage CPJ-hUC cells. They displayed a greater proliferation limit and a higher degree of multi-lineage differentiation in vitro and warrant further investigation to determine their full differentiation capacity, and therapeutic and regenerative medicine potential.

BET protein inhibitor JQ1 inhibits growth and modulates WNT signaling in mesenchymal stem cells.

BACKGROUND: Efficacy and safety of anticancer drugs are traditionally studied using cancer cell lines and animal models. The thienodiazepine class of BET inhibitors, such as JQ1, has been extensively studied for the potential treatment of hematological malignancies and several small molecules belonging to this class are currently under clinical investigation. While these compounds are well known to inhibit cancer cell growth and cause apoptosis, their effects on stem cells, particularly mesenchymal stem cells (MSCs), which are important for regeneration of damaged cells and tissues, are unknown. In this study we employed umbilical cord derived MSCs as a model system to evaluate the safety of JQ1. METHODS: Cord derived MSCs were treated with various doses of JQ1 and subjected to cell metabolic activity, apoptosis, and cell cycle analyses using MTT assay, Annexin-V/FITC and PI staining, and flow cytometry, respectively. The effect of JQ1 on gene expression was determined using microarray and quantitative real-time reverse transcriptase polymerase chain reaction analysis. Furthermore, protein expression of apoptotic and neuronal markers was carried out using western blot and immunostaining, respectively. RESULTS: Our results showed that JQ1 inhibited cell growth and caused cell cycle arrest in G1 phase but did not induce apoptosis or senescence. JQ1 also down-regulated genes involved in self-renewal, cell cycle, DNA replication, and mitosis, which may have negative implications on the regenerative potential of MSCs. In addition, JQ1 interfered with signaling pathways by down regulating the expression of WNT, resulting in limiting the self-renewal. These results suggest that anticancer agents belonging to the thienodiazepine class of BET inhibitors should be carefully evaluated before their use in cancer therapy. CONCLUSIONS: This study revealed for the first time that JQ1 adversely affected MSCs, which are important for repair and regeneration. JQ1 specifically modulated signal transduction and inhibited growth as well as self-renewal. These findings suggest that perinatal MSCs could be used to supplement animal models for investigating the safety of anticancer agents and other drugs.

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells.

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging. METHODS: Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers. RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively. CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.