Showing papers in "Stem Cells in 2013"

An Efficient Nonviral Method to Generate Integration-Free Human-Induced Pluripotent Stem Cells from Cord Blood and Peripheral Blood Cells†‡§

Abstract: The generation of induced pluripotent stem cells (iPSCs) provides the opportunity to use patient-specific somatic cells, which are a valuable source for disease modeling and drug discovery. To promote research involving these cells, it is important to make iPSCs from easily accessible and less invasive tissues, like blood. We have recently reported the efficient generation of human iPSCs from adult fibroblasts using a combination of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53. We herein report a modified protocol enabling efficient iPSC induction from CD34+ cord blood cells and from peripheral blood isolated from healthy donors using these plasmid vectors. The original plasmid mixture could induce iPSCs; however, the efficiency was low. The addition of EBNA1, an essential factor for episomal amplification of the vectors, by an extra plasmid greatly increased the efficiency of iPSC induction, especially when the induction was performed from αβT cells. This improvement enabled the establishment of blood-derived iPSCs from seven healthy donors ranging in age from their 20s to their 60s. This induction method will be useful for the derivation of patient-specific integration-free iPSCs and would also be applicable to the generation of clinical-grade iPSCs in the future.

MiR-133b promotes neural plasticity and functional recovery after treatment of stroke with multipotent mesenchymal stromal cells in rats via transfer of exosome-enriched extracellular particles

Abstract: To test, in vivo, the hypothesis that exosomes from multipotent mesenchymal stromal cells (MSCs) mediate microRNA 133b (miR-133b) transfer which promotes neurological recovery from stroke, we used knockin and knockdown technologies to upregulate or downregulate the miR-133b level in MSCs (miR-133b(+) MSCs or miR-133b(-) MSCs) and their corresponding exosomes, respectively. Rats were subjected to middle cerebral artery occlusion (MCAo) and were treated with naive MSCs, miR-133b(+) MSCs, or miR-133b(-) MSC at 1 day after MCAo. Compared with controls, rats receiving naive MSC treatment significantly improved functional recovery and exhibited increased axonal plasticity and neurite remodeling in the ischemic boundary zone (IBZ) at day 14 after MCAo. The outcomes were significantly enhanced with miR-133b(+) MSC treatment, and were significantly decreased with miR-133b(-) MSC treatment, compared to naive MSC treatment. The miR-133b level in exosomes collected from the cerebral spinal fluid was significantly increased after miR-133b(+) MSC treatment, and was significantly decreased after miR-133b(-) MSC treatment at day 14 after MCAo, compared to naive MSC treatment. Tagging exosomes with green fluorescent protein demonstrated that exosomes-enriched extracellular particles were released from MSCs and transferred to adjacent astrocytes and neurons. The expression of selective targets for miR-133b, connective tissue growth factor and ras homolog gene family member A, was significantly decreased in the IBZ after miR-133b(+) MSC treatment, while their expression remained at similar elevated levels after miR-133b(-) MSC treatment, compared to naive MSC treatment. Collectively, our data suggest that exosomes from MSCs mediate the miR-133b transfer to astrocytes and neurons, which regulate gene expression, subsequently benefit neurite remodeling and functional recovery after stroke.

Multipotent Stromal Cells Induce Human Regulatory T Cells Through a Novel Pathway Involving Skewing of Monocytes Toward Anti-inflammatory Macrophages

Abstract: Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture-expanded bone marrow-derived MSC promote the generation of CD4+CD25hiFoxP3+ T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC-conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF-β1 mediates induction of Tregs. TGF-β1 is constitutively secreted by MSC, suggesting that the MSC-induced generation of Tregs by TGF-β1 was independent of the interaction between MSC and PBMC. Monocyte-depletion studies showed that monocytes are indispensable for MSC-induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL-10 and CCL-18, which is mediated by as yet unidentified MSC-derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF-β1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC. Stem Cells 2013;31:1980-1991

Concise Review: Maturation Phases of Human Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract: Human pluripotent stem cell-derived cardiomyocytes (hPS-CM) may offer a number of advantages over previous cardiac models, however, questions of their immaturity complicate their adoption as a new in vitro model. hPS-CM differ from adult cardiomyocytes with respect to structure, proliferation, metabolism and electrophysiology, better approximating fetal cardiomyocytes. Time in culture appears to significantly impact phenotype, leading to what can be referred to as early and late hPS-CM. This work surveys the phenotype of hPS-CM, including structure, bioenergetics, sensitivity to damage, gene expression, and electrophysiology, including action potential, ion channels, and intracellular calcium stores, while contrasting fetal and adult CM with hPS-CM at early and late time points after onset of differentiation.

Differentially Activated Macrophages Orchestrate Myogenic Precursor Cell Fate During Human Skeletal Muscle Regeneration

Abstract: Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here, we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory MPs inhibited MPC fusion while anti-inflammatory MPs strongly promoted MPC differentiation by increasing their commitment into differentiated myocytes and the formation of mature myotubes. Furthermore, the in vivo time course of expression of myogenic and MP markers was studied in regenerating human healthy muscle after damage. We observed that regenerating areas containing proliferating MPCs were preferentially associated with MPs expressing proinflammatory markers. In the same muscle, regenerating areas containing differentiating myogenin-positive MPCs were preferentially coupled to MPs harboring anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates tissue repair.

Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.

Abstract: Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive), endocrine precursors (NKX2.2/synaptophysin-positive, hormone/NKX6.1-negative), and polyhormonal cells (insulin/glucagon-positive, NKX6.1-negative). However, the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional β-cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1-expressing cells (∼85%–90%) but were distinguished by their relatively high (∼80%) or low (∼25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study, but only NKX6.1-high grafts displayed robust meal-, glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells, whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm-enriched population can mature into highly functional β-cells with only a minor contribution from the endocrine subpopulation. Stem Cells 2013;31:2432–2442

Multipotential differentiation of human urine‐derived stem cells: Potential for therapeutic applications in urology

Abstract: We sought to biologically characterize and identify a subpopulation of urine-derived stem cells (USCs) with the capacity for multipotent differentiation. We demonstrated that single USCs can expand to a large population with 60-70 population doublings. Nine of 15 individual USC clones expressed detectable levels of telomerase and have long telomeres. These cells expressed pericyte and mesenchymal stem cell markers. Upon induction with appropriate media in vitro, USCs differentiated into bladder-associated cell types, including functional urothelial and smooth muscle cell lineages. When the differentiated USCs were seeded onto a scaffold and subcutaneously implanted into nude mice, multilayered tissue-like structures formed consisting of urothelium and smooth muscle. Additionally, USCs were able to differentiate into endothelial, osteogenic, chondrogenic, adipogenic, skeletal myogenic, and neurogenic lineages but did not form teratomas during the 1-month study despite telomerase activity. USCs may be useful in cell-based therapies and tissue engineering applications, including urogenital reconstruction.

Autologous adipose tissue‐derived stem cells treatment demonstrated favorable and sustainable therapeutic effect for Crohn's fistula

Abstract: Fistula is a representative devastating complication in Crohn's patients due to refractory to conventional therapy and high recurrence. In our phase I clinical trial, adipose tissue-derived stem cells (ASCs) demonstrated their safety and therapeutic potential for healing fistulae associated with Crohn's disease. This study was carried out to evaluate the efficacy and safety of ASCs in patients with Crohn's fistulae. In this phase II study, forty-three patients were treated with ASCs. The amount of ASCs was proportioned to fistula size and fistula tract was filled with ASCs in combination with fibrin glue after intralesional injection of ASCs. Patients without complete closure of fistula at 8 weeks received a second injection of ASCs containing 1.5 times more cells than the first injection. Fistula healing at week 8 after final dose injection and its sustainability for 1-year were evaluated. Healing was defined as a complete closure of external opening without any sign of drainage and inflammation. A modified per-protocol analysis showed that complete fistula healing was observed in 27/33 patients (82%) by 8 weeks after ASC injection. Of 27 patients with fistula healing, 26 patients completed additional observation study for 1-year and 23 patients (88%) sustained complete closure. There were no adverse events related to ASC administration. ASC treatment for patients with Crohn's fistulae was well tolerated, with a favorable therapeutic outcome. Furthermore, complete closure was well sustained. These results strongly suggest that autologous ASC could be a novel treatment option for the Crohn's fistula with high-risk of recurrence.

Concise Review: The Evolution of human pluripotent stem cell culture: From feeder cells to synthetic coatings†‡§

Abstract: Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells and embryonic stem cells, in an undifferentiated state typically depend on the support of feeder cells such as mouse embryonic fibroblasts (MEFs) or an extracellular matrix such as Matrigel. Culture conditions that depend on these undefined support systems limit our ability to interpret mechanistic studies aimed at resolving how hPSCs interact with their extracellular environment to remain in a unique undifferentiated state and to make fate-changing lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel ultimately hinder our ability to use pluripotent stem cells to treat debilitating human diseases. Many of these obstacles have been overcome by the development of synthetic coatings and bioreactors that support hPSC expansion and self-renewal within defined culture conditions that are free from xenogeneic contamination. The establishment of defined culture conditions and synthetic matrices will facilitate studies to more precisely probe the molecular basis of pluripotent stem cell self-renewal and differentiation. When combined with three-dimensional cultures in bioreactors, these systems will also enable large-scale expansion for future clinical applications. STEM CELLS 2013;31:1–7

Efficient Generation of Astrocytes from Human Pluripotent Stem Cells in Defined Conditions

Abstract: Astrocytes can be generated from various tissue sources including human pluripotent stem cells (PSC). In this manuscript, we describe a chemically defined xeno-free medium culture system for rapidly generating astrocytes from neural stem cells derived from PSC. We show that astrocyte development in vitro, mimics normal development in vivo, and also passes through a CD44(+) astrocyte precursor stage. Astrocytes generated by our method display similar gene expression patterns, morphological characteristics and functional properties to primary astrocytes, and they survive and integrate after xenotransplantation. Whole genome expression profiling of astrocyte differentiation was performed at several time points of differentiation, and the results indicate the importance of known regulators and identify potential novel regulators and stage-specific lineage markers.

Tumor-Associated Macrophages Regulate Murine Breast Cancer Stem Cells Through a Novel Paracrine EGFR/Stat3/Sox-2 Signaling Pathway†‡§

Abstract: The cancer stem cell (CSC) hypothesis has gained significant recognition as a descriptor of tumorigenesis. Additionally, tumor-associated macrophages (TAMs) are known to promote growth and metastasis of breast cancer. However, it is not known whether TAMs mediate tumorigenesis through regulation of breast CSCs. Here, we report that TAMs promote CSC-like phenotypes in murine breast cancer cells by upregulating their expression of Sox-2. These CSC-like phenotypes were characterized by increased Sox-2, Oct-4, Nanog, AbcG2, and Sca-1 gene expression, in addition to increased drug-efflux capacity, resistance to chemotherapy, and increased tumorigenicity in vivo. Downregulation of Sox-2 in tumor cells by siRNA blocked the ability of TAMs to induce these CSC-like phenotypes and inhibited tumor growth in vivo. Furthermore, we identified a novel epidermal growth factor receptor (EGFR)/signal transducers and activators of transcription 3 (Stat3)/Sox-2 paracrine signaling pathway between macrophages and mouse breast cancer cells that is required for macrophage-induced upregulation of Sox-2 and CSC phenotypes in tumor cells. We showed that this crosstalk was effectively blocked by the small molecule inhibitors AG1478 or CDDO-Im against EGFR and Stat3, respectively. Therefore, our report identifies a novel role for TAMs in breast CSC regulation and establishes a rationale for targeting the EGFR/Stat3/Sox-2 signaling pathway for CSC therapy.

Human Pericytes for Ischemic Heart Repair

Abstract: Human microvascular pericytes (CD146(+)/34(-)/45(-)/56(-)) contain multipotent precursors and repair/regenerate defective tissues, notably skeletal muscle. However, their ability to repair the ischemic heart remains unknown. We investigated the therapeutic potential of human pericytes, purified from skeletal muscle, for treating ischemic heart disease and mediating associated repair mechanisms in mice. Echocardiography revealed that pericyte transplantation attenuated left ventricular dilatation and significantly improved cardiac contractility, superior to CD56+ myogenic progenitor transplantation, in acutely infarcted mouse hearts. Pericyte treatment substantially reduced myocardial fibrosis and significantly diminished infiltration of host inflammatory cells at the infarct site. Hypoxic pericyte-conditioned medium suppressed murine fibroblast proliferation and inhibited macrophage proliferation in vitro. High expression by pericytes of immunoregulatory molecules, including interleukin-6, leukemia inhibitory factor, cyclooxygenase-2, and heme oxygenase-1, was sustained under hypoxia, except for monocyte chemotactic protein-1. Host angiogenesis was significantly increased. Pericytes supported microvascular structures in vivo and formed capillary-like networks with/without endothelial cells in three-dimensional cocultures. Under hypoxia, pericytes dramatically increased expression of vascular endothelial growth factor-A, platelet-derived growth factor-β, transforming growth factor-β1 and corresponding receptors while expression of basic fibroblast growth factor, hepatocyte growth factor, epidermal growth factor, and angiopoietin-1 was repressed. The capacity of pericytes to differentiate into and/or fuse with cardiac cells was revealed by green fluorescence protein labeling, although to a minor extent. In conclusion, intramyocardial transplantation of purified human pericytes promotes functional and structural recovery, attributable to multiple mechanisms involving paracrine effects and cellular interactions.

Improved Cell Therapy Protocols for Parkinson's Disease Based on Differentiation Efficiency and Safety of hESC-, hiPSC-, and Non-Human Primate iPSC-Derived Dopaminergic Neurons†

Abstract: The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.

CD133 is essential for glioblastoma stem cell maintenance.

Abstract: The role of the cell surface CD133 as a cancer stem cell marker in glioblastoma (GBM) has been widely investigated, since it identifies cells that are able to initiate neurosphere growth and form heterogeneous tumors when transplanted in immune-compromised mice. However, evidences of CD133-negative cells exhibiting similar properties have also been reported. Moreover, the functional role of CD133 in cancer stem/progenitor cells remains poorly understood. We studied the biological effects of CD133 downregulation in GBM patient-derived neurospheres. Our results indicate that there is not a hierarchical relation between CD133-positive and CD133-negative cells composing the neurospheres. Indeed, CD133 appears in an interconvertible state, changing its subcellular localization between the cytoplasm and the plasmamembrane of neurosphere cells. Silencing of CD133 in human GBM neurospheres using lentivirus-mediated short hairpin RNA impairs the self-renewal and tumorigenic capacity of neurosphere cells. These results imply that CD133 could be used as a therapeutic target in GBMs.

Concise review: two negative feedback loops place mesenchymal stem/stromal cells at the center of early regulators of inflammation.

Abstract: Recent data demonstrated that MSCs can be activated by proinflammatory signals to introduce two negative feedback loops into the generic pathway of inflammation. In one loop, the activated MSCs secrete PGE2 that drives resident macrophages with an M1 proinflammatory phenotype toward an M2 anti-inflammatory phenotype. In the second loop, the activated MSCs secrete TSG-6 that interacts with CD44 on resident macrophages to decrease TLR2/NFκ-B signaling and thereby decrease the secretion of proinflammatory mediators of inflammation. The PGE2 and TSG-6 negative feedback loops allow MSCs to serve as regulators of the very early phases of inflammation. These and many related observations suggest that the MSC-like cells found in most tissues may be part of the pantheon of cells that protect us from foreign invaders, tissue injury, and aging.

Human bone marrow-derived mesenchymal stem cells suppress human glioma growth through inhibition of angiogenesis.

Abstract: Tumor tropism of human bone marrow-derived mesenchymal stem cells (MSC) has been exploited for the delivery of therapeutic genes for anticancer therapy. However, the exact contribution of these cells in the tumor microenvironment remains unknown. In this study, we examined the biological effect of MSC on tumor cells. The results showed that MSC inhibited the growth of human glioma cell lines and patient-derived primary glioma cells in vitro. Coadministration of MSC and glioma cells resulted in significant reduction in tumor volume and vascular density, which was not observed when glioma was injected with immortalized normal human astrocytes. Using endothelial progenitor cells (EPC) from healthy donors and HUVEC endothelial cells, the extent of EPC recruitment and capacity to form endothelial tubes was significantly impaired in conditioned media derived from MSC/glioma coculture, suggesting that MSC suppressed tumor angiogenesis through the release of antiangiogenic factors. Further studies using antibody array showed reduced expression of platelet-derived growth factor (PDGF)-BB and interleukin (IL)-1β in MSC/glioma coculture when compared with controls. In MSC/glioma coculture, PDGF-BB mRNA and the corresponding proteins (soluble and membrane bound forms) as well as the receptors were found to be significantly downregulated when compared with that of glioma cocultured with normal human astrocytes or glioma monoculture. Furthermore, IL-1β, phosphorylated Akt, and cathepsin B proteins were also reduced in MSC/glioma. Taken together, these data indicated that the antitumor effect of MSC may be mediated through downregulation of PDGF/PDGFR axis, which is known to play a key role in glioma angiogenesis. STEM Cells2013;31:146–155

Brief Report: The lincRNA Hotair Is Required for Epithelial-to-Mesenchymal Transition and Stemness Maintenance of Cancer Cell Lines

Abstract: Hotair is a member of the recently described class of noncoding RNAs called lincRNA (large intergenic noncoding RNA). Various studies suggest that Hotair acts regulating epigenetic states by recruiting chromatin-modifying complexes to specific target sequences that ultimately leads to suppression of several genes. Although Hotair has been associated with metastasis and poor prognosis in different tumor types, a deep characterization of its functions in cancer is still needed. Here, we investigated the role of Hotair in the scenario of epithelial-to-mesenchymal transition (EMT) and in the arising and maintenance of cancer stem cells (CSCs). We found that treatment with TGF-β1 resulted in increased Hotair expression and triggered the EMT program. Interestingly, ablation of Hotair expression by siRNA prevented the EMT program stimulated by TGF-β1, and also the colony-forming capacity of colon and breast cancer cells. Furthermore, we observed that the colon CSC subpopulation (CD133(+)/CD44(+)) presents much higher levels of Hotair when compared with the non-stem cell subpopulation. These results indicate that Hotair acts as a key regulator that controls the multiple signaling mechanisms involved in EMT. Altogether, our data suggest that the role of Hotair in tumorigenesis occurs through EMT triggering and stemness acquisition.

Umbilical cord blood therapy potentiated with erythropoietin for children with cerebral palsy: a double-blind, randomized, placebo-controlled trial.

Abstract: Allogeneic umbilical cord blood (UCB) has therapeutic potential for cerebral palsy (CP). Concomitant administration of recombinant human erythropoietin (rhEPO) may boost the efficacy of UCB, as it has neurotrophic effects. The objectives of this study were to assess the safety and efficacy of allogeneic UCB potentiated with rhEPO in children with CP. Children with CP were randomly assigned to one of three parallel groups: the pUCB group, which received allogeneic UCB potentiated with rhEPO; the EPO group, which received rhEPO and placebo UCB; and the Control group, which received placebo UCB and placebo rhEPO. All participants received rehabilitation therapy. The main outcomes were changes in scores on the following measures during the 6 months treatment period: the gross motor performance measure (GMPM), gross motor function measure, and Bayley scales of infant development-II (BSID-II) Mental and Motor scales (18). F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET/CT) and diffusion tensor images (DTI) were acquired at baseline and followed up to detect changes in the brain. In total, 96 subjects completed the study. Compared with the EPO (n = 33) and Control (n = 32) groups, the pUCB (n = 31) group had significantly higher scores on the GMPM and BSID-II Mental and Motor scales at 6 months. DTI revealed significant correlations between the GMPM increment and changes in fractional anisotropy in the pUCB group. 18F-FDG-PET/CT showed differential activation and deactivation patterns between the three groups. The incidence of serious adverse events did not differ between groups. In conclusion, UCB treatment ameliorated motor and cognitive dysfunction in children with CP undergoing active rehabilitation, accompanied by structural and metabolic changes in the brain. Stem Cells2013;31:581–591

MELK-dependent FOXM1 phosphorylation is essential for proliferation of glioma stem cells.

Abstract: Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM.

Dynamic compaction of human mesenchymal stem/precursor cells into spheres self-activates caspase-dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6, and STC1)

Abstract: Human mesenchymal stem/precursor cells (MSC) are similar to some other stem/progenitor cells in that they compact into spheres when cultured in hanging drops or on nonadherent surfaces. Assembly of MSC into spheres alters many of their properties, including enhanced secretion of factors that mediate inflammatory and immune responses. Here we demonstrated that MSC spontaneously aggregated into sphere-like structures after injection into a subcutaneous air pouch or the peritoneum of mice. The structures were similar to MSC spheres formed in cultures demonstrated by the increased expression of genes for inflammation-modulating factors TSG6, STC1, and COX2, a key enzyme in production of PGE2. To identify the signaling pathways involved, hanging drop cultures were used to follow the time-dependent changes in the cells as they compacted into spheres. Among the genes upregulated were genes for the stress-activated signaling pathway for IL1α/β, and the contact-dependent signaling pathway for Notch. An inhibitor of caspases reduced the upregulation of IL1A/B expression, and inhibitors of IL1 signaling decreased production of PGE2, TSG6, and STC1. Also, inhibition of IL1A/B expression and secretion of PGE2 negated the anti-inflammatory effects of MSC spheres on stimulated macrophages. Experiments with γ-secretase inhibitors suggested that Notch signaling was also required for production of PGE2 but not TSG6 or STC1. The results indicated that assembly of MSC into spheres triggers caspase-dependent IL1 signaling and the secretion of modulators of inflammation and immunity. Similar aggregation in vivo may account for some of the effects observed with administration of the cells in animal models.

Mesenchymal stromal cells improve survival during sepsis in the absence of heme oxygenase-1: the importance of neutrophils

Abstract: The use of mesenchymal stromal cells (MSCs) for treatment of bacterial infections, including systemic processes like sepsis, is an evolving field of investigation. This study was designed to investigate the potential use of MSCs, harvested from compact bone, and their interactions with the innate immune system, during polymicrobial sepsis induced by cecal ligation and puncture (CLP). We also wanted to elucidate the role of endogenous heme oxygenase (HO)-1 in MSCs during a systemic bacterial infection. MSCs harvested from the bones of HO-1 deficient (-/-) and wild-type (+/+) mice improved the survival of HO-1(-/-) and HO-1(+/+) recipient mice when administered after the onset of polymicrobial sepsis induced by CLP, compared with the administration of fibroblast control cells. The MSCs, originating from compact bone in mice, enhanced the ability of neutrophils to phagocytize bacteria in vitro and in vivo and to promote bacterial clearance in the peritoneum and blood after CLP. Moreover, after depleting neutrophils in recipient mice, the beneficial effects of MSCs were entirely lost, demonstrating the importance of neutrophils for this MSC response. MSCs also decreased multiple organ injury in susceptible HO-1(-/-) mice, when administered after the onset of sepsis. Taken together, these data demonstrate that the beneficial effects of treatment with MSCs after the onset of polymicrobial sepsis is not dependent on endogenous HO-1 expression, and that neutrophils are crucial for this therapeutic response.

Dysfunction of endothelial progenitor cells from smokers and chronic obstructive pulmonary disease patients due to increased dna damage and senescence

Abstract: Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in those with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples from healthy nonsmokers, healthy smokers, and COPD patients. BOEC from smokers and COPD patients showed increased DNA double-strand breaks and senescence compared to nonsmokers. Senescence negatively correlated with the expression and activity of sirtuin-1 (SIRT1), a protein deacetylase that protects against DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia mutated (ATM) kinase resulted in upregulation of SIRT1 expression and decreased senescence. Treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or an ATM inhibitor (KU-55933) also rescued the senescent phenotype. Using an in vivo mouse model of angiogenesis, we demonstrated that senescent BOEC from COPD patients are dysfunctional, displaying impaired angiogenic ability and increased apoptosis compared to cells from healthy nonsmokers. Therefore, this study identifies epigenetic regulation of DNA damage and senescence as pathogenetic mechanisms linked to endothelial progenitors' dysfunction in smokers and COPD patients. These defects may contribute to vascular disease and cardiovascular events in smokers and could therefore constitute therapeutic targets for intervention.

Mesenchymal stem cells and endothelial progenitor cells decrease renal injury in experimental swine renal artery stenosis through different mechanisms

Abstract: Endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) augment tissue repair but possess slightly different properties. How the cellular phenotype affects the efficacy of this approach in renovascular disease is incompletely understood. This study tested the hypothesis that EPC and MSC protect the poststenotic kidney by blunting different disease pathways. Peripheral blood EPC and adipose-derived MSC were expanded and characterized by cell surface markers (e.g., CD34/kinase insert domain receptor, or CD44/CD90). Single-kidney hemodynamics and function were assessed in pigs after 10 weeks of renal artery stenosis (RAS) treated 4 weeks earlier with an intrarenal infusion of vehicle (n = 7), EPC (RAS+EPC) or MSC (RAS+MSC) (both 10 × 10(6), n = 6), and normal controls (n = 7). Kidney disease mechanisms were evaluated ex vivo. The ability of EPC and MSC to attenuate endoplasmic reticulum (ER) stress was also studied in isolated ER and in tubular cells cocultured with EPC and MSC. Glomerular filtration rate in RAS was lower than controls, increased in RAS+EPC, and further improved in RAS+MSC, although both improved renal blood flow similarly. EPC prominently enhanced renal growth factor expression and decreased oxidative stress, while MSC more significantly attenuated renal inflammation, ER stress, and apoptosis. Furthermore, MSC induced a greater decrease in caspase-3 and CHOP expression in cultured tubular cells through mechanisms involving cell contact. EPC and MSC achieve a comparable decrease of kidney injury in RAS by different mechanisms, although MSC elicited slightly superior improvement of renal function. These results support development of cell-based approaches for management of renovascular disease and suggest cell selection based on the underlying pathophysiology of kidney injury.

Integration-Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology

Abstract: Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.

Late Outgrowth Endothelial Cells Resemble Mature Endothelial Cells and Are Not Derived from Bone Marrow

Abstract: A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte-colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD34 1 CD133 2 CD146 1 cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real-time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 6 2.7 vs. 6.6 6 3.7 vessels, respectively, n 5 9). Bone marrow-derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34 1 CD133 2 CD146 1 mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrowderived circulating precursor for endothelial cells. STEM CELLS 2013;31:338–348

Brief Report: Intravital Imaging of Cancer Stem Cell Plasticity in Mammary Tumors

Abstract: It is widely debated whether all tumor cells in mammary tumors have the same potential to propagate and maintain tumor growth or whether there is a hierarchical organization. Evidence for the latter theory is mainly based on the ability or failure of transplanted tumor cells to produce detectable tumors in mice with compromised immune systems; however, this assay has lately been disputed to accurately reflect cell behavior in unperturbed tumors. Lineage tracing experiments have recently shown the existence of a small population of cells, referred to as cancer stem cells (CSCs), that maintains and provides growth of squamous skin tumors and intestinal adenomas. However, the lineage tracing techniques used in these studies provide static images and lack the ability to study whether stem cell properties can be obtained or lost, a process referred to as stem cell plasticity. Here, by intravital lineage tracing, we report for the first time the existence of CSCs in unperturbed mammary tumors and demonstrate CSC plasticity. Our data indicate that existing CSCs disappear and new CSCs form during mammary tumor growth, illustrating the dynamic nature of these cells. Stem Cells 2013;31:602–606

MiRNA-20 and mirna-106a regulate spermatogonial stem cell renewal at the post-transcriptional level via targeting STAT3 and Ccnd1

Abstract: Studies on spermatogonial stem cells (SSCs) are of unusual significance because they are the unique stem cells that transmit genetic information to subsequent generations and they can acquire pluripotency to become embryonic stem-like cells that have therapeutic applications in human diseases. MicroRNAs (miRNAs) have recently emerged as critical endogenous regulators in mammalian cells. However, the function and mechanisms of individual miRNAs in regulating SSC fate remain unknown. Here, we report for the first time that miRNA-20 and miRNA-106a are preferentially expressed in mouse SSCs. Functional assays in vitro and in vivo using miRNA mimics and inhibitors reveal that miRNA-20 and miRNA-106a are essential for renewal of SSCs. We further demonstrate that these two miRNAs promote renewal at the post-transcriptional level via targeting STAT3 and Ccnd1 and that knockdown of STAT3, Fos, and Ccnd1 results in renewal of SSCs. This study thus provides novel insights into molecular mechanisms regulating renewal and differentiation of SSCs and may have important implications for regulating male reproduction.

Concise review: Adult salivary gland stem cells and a potential therapy for xerostomia.

Abstract: The ability to speak, swallow, masticate, taste food, and maintain a healthy oral cavity is heavily reliant on the presence of saliva, the hugely important effect of which on our everyday lives is often unappreciated. Hyposalivation, frequently experienced by people receiving radiation therapy for head and neck cancers, results in a plethora of symptoms whose combined effect can drastically reduce quality of life. Although artificial lubricants and drugs stimulating residual function are available to ameliorate the consequences of hyposalivation, their effects are at best transient. Such management techniques do not address the source of the problem: a lack of functional saliva-producing acinar cells, resulting from radiation-induced stem cell sterilization. Post-radiotherapy stimulation of cell proliferation only results in improved saliva secretion when part of the tissue has been spared or when the dose to the salivary gland (SG) remains below a certain level. Therefore, stem cell replacement therapy may be a good option to treat radiation-induced hyposalivation. Substantial progress has been made lately in the understanding of cell turnover in the SG, and the recent identification of stem and progenitor cell populations in the SG provides a basis for studies toward development of a stem cell-based therapy for xerostomia. Here, we review the current state of knowledge of SG stem cells and their potential for use in a cell-based therapy that may provide a more durable cure for hyposalivation. STEM CELLS 2013;31:613–619

MicroRNA-302 increases reprogramming efficiency via repression of NR2F2

Abstract: MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302, which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as "KMOS3") when compared to using four factors ("KMOS"). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.

Concise review: adult mesenchymal stromal cell therapy for inflammatory diseases: how well are we joining the dots?

Abstract: Mesenchymal stromal (stem) cells (MSCs) continue to be a strong area of focus for academic- and industry-based researchers who share the goal of expanding their therapeutic use for diverse inflammatory and immune-mediated diseases. Recently, there has been an accelerated rate of scientific publication, clinical trial activity, and commercialisation in the field. This has included the reporting of exciting new developments in four areas that will be of key importance to future successful use of MSC-based therapies in large numbers of patients: (a) fundamental biology of the primary cells in bone marrow and other tissues that give rise to MSCs in culture. (b) Mechanisms by which MSCs modulate immune and inflammatory responses in vivo. (c) Insights into MSC kinetics, safety, and efficacy in relevant animal disease models. (d) Isolation, definition, and clinical trial-based testing of human MSCs by biomedical companies and academic medical centers. Despite this progress, it remains unclear whether MSCs will enter mainstream therapeutic practice as a frequently used alternative to pharmacotherapy or surgical/radiological procedures in the foreseeable future. In this review, we summarize some of the most significant new developments for each of the four areas that contribute to the process of translating MSC research to the clinical arena. In the context of this recent progress, we discuss key challenges and specific knowledge gaps which, if not addressed in a coordinated fashion, may hinder the creation of robust "translational pipelines" for consolidating the status of MSC-based therapies.