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.

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.

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.

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.

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.

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.

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.