Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells

We examined the gene expression and DNA methylation of 49 human induced pluripotent stem cells (hiPSCs) and 10 human embryonic stem cells and found overlapped variations in gene expression and DNA methylation in the two types of human pluripotent stem cell lines. Comparisons of the in vitro neural differentiation of 40 hiPSCs and 10 human embryonic stem cells showed that seven hiPSC clones retained a significant number of undifferentiated cells even after neural differentiation culture and formed teratoma when transplanted into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression levels of several genes, including those expressed from long terminal repeats of specific human endogenous retroviruses. These data demonstrated a subset of hiPSC lines that have aberrant gene expression and defective potential in neural differentiation, which need to be identified and eliminated before applications in regenerative medicine.Human pluripotent stem cells possess a robust potential for proliferation and provide useful sources of cells for regenerative medicine and drug discovery. Two types of human pluripotent stem cells have been generated: human embryonic stem cells (hESCs) derived from blastocysts (1) and induced pluripotent stem cells (hiPSCs), which are generated from somatic cells by factor-mediated reprogramming (2, 3).In the past few years, findings have been controversial in regard to whether hESCs and hiPSCs are distinct cell types. Some reports have argued that they could not be clearly distinguished (4–6), whereas others have reported that they have differences in their gene expression (7–10), DNA methylation (10–13), and capacity for differentiation (14). In the latter papers, relatively small numbers of cell lines were generally compared. In addition, most comparisons used pluripotent cell lines from various laboratories, so the observed differences may be attributable to laboratory-specific variations owing to technical differences (15).To overcome these problems, we compared the mRNA and microRNA (miRNA) expression and DNA methylation between 10 hESCs and 49 hiPSCs that had been cultured under the same conditions. Furthermore, we compared the in vitro directed neural differentiation of these pluripotent stem cells.     

Novel recurrently mutated genes in African American colon cancers

We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.Colorectal cancer (CRC) is a leading cause of cancer mortality world-wide. CRC incidence and mortality rates are both increased in African Americans (AA) compared with Caucasians Americans (1–3). Although several factors likely play a role, the contribution of potential differences in tumor genetics to this disparity have yet to be fully explored (1, 3). In particular, AA CRCs were notably underrepresented in the four major published CRC sequencing studies (4–7), accounting for only two annotated AA cases of the 333 total CRCs studied (4–7). Accordingly, we initiated this study to compare the mutational landscapes of CRCs from AA individuals versus Caucasians.     

Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson’s disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC–DAn) in culture. Here, we showed that after the striatal transplantation of pNSC–DAn, (i) pNSC–DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (and not from injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra pars compacta and their projecting axons, resulting in loss of dopamine (DA) release in the striatum (1). During the last two decades, cell-replacement therapy has proven, at least experimentally, to be a potential treatment for PD patients (2–7) and in animal models (8–15). The basic principle of cell therapy is to restore the DA release by transplanting new DA-like cells. Until recently, obtaining enough transplantable cells was a major bottleneck in the practicability of cell therapy for PD. One possible source is embryonic stem cells (ESCs), which can develop infinitely into self-renewable pluripotent cells with the potential to generate any type of cell, including DA neurons (DAns) (16, 17).Recently, several groups including us have introduced rapid and efficient ways to generate primitive neural stem cells (pNSCs) from human ESCs using small-molecule inhibitors under chemically defined conditions (12, 18, 19). These cells are nonpolarized neuroepithelia and retain plasticity upon treatment with neuronal developmental morphogens. Importantly, pNSCs differentiate into DAns (pNSC–DAn) with high efficiency (∼65%) after patterning by sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) in vitro, providing an immediate and renewable source of DAns for PD treatment. Importantly, the striatal transplantation of human ESC-derived DA-like neurons, including pNSC–DAn, are able to relieve the motor defects in a PD rat model (11–13, 15, 19–23). Before attempting clinical translation of pNSC–DAn, however, there are two fundamental open questions. (i) Can pNSC–DAn functionally restore the striatal DA levels in vivo? (ii) What cells release the restored DA, pNSC–DAn themselves or resident neurons/cells repaired by the transplants?Regarding question 1, a recent study using nafion-coated carbon fiber electrodes (CFEs) reported that the amperometric current is rescued in vivo by ESC (pNSC–DAn-like) therapy (19). Both norepinephrine (NE) and serotonin are present in the striatum (24, 25). However, CFE amperometry/chronoamperometry alone cannot distinguish DA from other monoamines in vivo, such as NE and serotonin (Fig. S1) (see also refs. 26–28). Considering that the compounds released from grafted ESC-derived cells are unknown, the work of Kirkeby et al. was unable to determine whether DA or other monoamines are responsible for the restored amperometric signal. Thus, the key question of whether pNSC–DAn can rescue DA release needs to be reexamined for the identity of the restored amperometric signal in vivo.Regarding question 2, many studies have proposed that DA is probably released from the grafted cells (8, 12, 13, 20), whereas others have proposed that the grafted stem cells might restore striatal DA levels by rescuing injured original cells (29, 30). Thus, whether the grafted cells are actually capable of synthesizing and releasing DA in vivo must be investigated to determine the future cellular targets (residual cells versus pNSC–DAn) of treatment.To address these two mechanistic questions, advanced in vivo methods of DA identification and DA recording at high spatiotemporal resolution are required. Currently, microdialysis-based HPLC (HPLC) (31–33) and CFE amperometric recordings (34, 35) have been used independently by different laboratories to assess evoked DA release from the striatum in vivo. The major advantage of microdialysis-based HPLC is to identify the substances secreted in the cell-grafted striatum (33), but its spatiotemporal resolution is too low to distinguish the DA release site (residual cells or pNSC–DAn). In contrast, the major advantage of CFE-based amperometry is its very high temporal (ms) and spatial (μm) resolution, making it possible to distinguish the DA release site (residual cells or pNSC–DAn) in cultured cells, brain slices, and in vivo (34–39), but it is unable to distinguish between low-level endogenous oxidizable substances (DA versus serotonin and NE) in vivo.In the present study, we developed a challenging experimental paradigm of combining the two in vivo methods, microdialysis-based HPLC and CFE amperometry, to identify the evoked substance as DA and its release site as pNSC–DAn in the striatum of PD rats.     

Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine. To overcome potential problems associated with transgene insertions, efforts have been directed over the past several years to generate transgene-free iPS cells by using non-viral-vector approaches. To date, however, cells generated through such procedures have had problems producing reproductively competent animals, suggesting that their quality needed further improvement. Here we report the use of optimized assemblies of reprogramming factors and selection markers incorporated into single plasmids as nonintegrating episomes to generate germ-line–competent iPS cells. In particular, the pMaster12 episome can produce transgene-free iPS cells that, when grown in 2i medium, recapitulate good mouse ES cells, in terms of their competency for generating germ-line chimeras.Although induced pluripotent stem (iPS) cells hold enormous promise for cell-based therapies (1, 2), their use in humans is still problematic because of their potential to also do harm to the patient. High among these risk factors is their potential to induce cancer. For example, use of viral vectors to randomly integrate genes into somatic cells used for human gene therapy trials has resulted in the induction of cancer in the recipient patients (3). Also, random integration of the exogenous reprograming genes in iPS cells increases tumor formation, morbidity, and mortality in mice generated from these cells (4, 5). Therefore, the safety and quality of iPS cells are of critical importance to their anticipated use for human cell-based therapies. To circumvent some of these safety issues, safer methods, especially nonviral methods, for the introduction of the reprogramming factors into somatic cells are being developed. These methods have included the use of plasmids (6, 7), the piggyBac (PB) transposon (8, 9), nonintegrating episomes (10–13), protein transduction (14, 15), transfection of mRNA and microRNAs (16–18), and small molecule inhibitors (19). Although these approaches have yielded transgene-free iPS cells, the competency of the resulting iPS cells to contribute to a functional germ line has not been adequately demonstrated.     

Numb-deficient satellite cells have regeneration and proliferation defects

The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury.Satellite cells represent a muscle-specific stem cell population that allows for muscle growth postnatally and is necessary for muscle repair (1). In response to muscle-fiber damage, quiescent satellite cells that lie along the myofibers under the plasmalemma are activated and proliferate. Proliferating satellite cells have a binary fate decision to make—they can differentiate into myoblasts and intercalate into myofibers by fusion to repair the damaged muscle or they can renew the satellite cell population and return to a quiescent state (2–4). Quiescent satellite cells express paired box 7 (Pax7), but low or undetectable levels of the myogenic regulatory factors Myf5 and MyoD (5, 6). Activated satellite cells robustly express Pax7 and MyoD/Myf5, but a subset will subsequently down-regulate the myogenic regulatory factors in the process of satellite cell self-renewal (7). Recent studies have demonstrated that, in vivo, Pax7-positive cells are necessary for muscle repair (8, 9).Notch signaling is an important regulator of satellite cell function; it is implicated in satellite cell activation, proliferation (2, 10, 11), and maintenance of quiescence (12, 13). Expression of constitutively active Notch1 results in maintenance of Pax7 expression and down-regulation of Myod/Myf5 whereas inhibition of Notch signaling leads to myogenic differentiation (10, 14). In fact, conditional ablation of Rbpj embryonically results in hypotrophic muscle (15), and, if ablated in the adult, satellite cells undergo spontaneous activation and precocious differentiation with a failure of self-renewal (12, 13). In adult muscle, the Notch ligand, Delta-like1 (Dll1), is expressed on satellite cells, myofibers, and newly differentiating myoblasts and is necessary for repair (10, 11, 16). In aged muscle, impairment of regeneration is due, in part, to a failure of Dll1 expression (17).Numb en`s four proteins with molecular masses of 65, 66, 71, and 72 kDa by alternative splicing of two exons (18, 19). The Numb proteins are cytoplasmic adaptors that direct ubiquitination and degradation of Notch1 by recruiting the E3 ubiquitin ligase Itch to the receptor (18–22). Numb is a cell-fate determinant that mediates asymmetric cell division, leading to selective Notch inhibition in one daughter cell and its subsequent differentiation whereas the other daughter has active Notch signaling and remains proliferative (10). Embryonically, Numb is expressed in the myotome whereas Notch1 is limited to the dermomyotome (23, 24). This pattern suggests that the expression of Numb in one daughter cell allows entry into the myogenic lineage. Indeed, overexpression of Numb embryonically increases the number of myogenic progenitors in the somite (25, 26).Numb expression increases during the activation and proliferative expansion of satellite cells, becoming asymmetrically segregated in transit-amplifying cells and leading to asymmetric cell divisions (10, 27). These observations led to a model in which Numb inhibits Notch signaling in one daughter satellite cell, allowing it to undergo myogenic differentiation. The molecular switch that controls the decision of satellite cell progeny to continue proliferating or to differentiate is not well understood. This process seems to be controlled by a decrease of Notch signaling due to increased expression of Numb and an increase in Wnt signaling (10–14, 17, 28). In these studies, we examined the role of Numb in satellite cell function by genetic deletion of Numb from myogenic progenitors and satellite cells. Our observations reveal that Numb is necessary for satellite cell-mediated repair. Furthermore, Numb-deficient satellite cells have an unexpected proliferation defect due to an up-regulation of Myostatin. These data indicate a unique role for Numb in regulating the activation and proliferation of satellite cells.     

Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling

Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.Harnessing the ability of adult stem cells to differentiate and contribute to tissue repair has enormous potential for wound healing, tissue regeneration, and restoration of organ functionality. However, controlling the fate of transplanted and/or endogenous progenitor cells to treat compromised tissues and organs remains a significant challenge (1, 2). Studies have shown that biomaterials recapitulating various physicochemical cues of the native tissue can be used to direct stem cell differentiation (3–9). Biomaterials-assisted transplantation of stem cells provides a promising approach to deliver cells to the targeted site and direct their differentiation to functional tissues. We and others have shown that biomaterials containing calcium phosphate (CaP) moieties, a major constituent of native bone tissue, can promote osteogenic differentiation of progenitor and stem cells and can facilitate in vivo bone tissue formation (10–20). However, to use CaP biomaterials efficiently for bone tissue repair, it is of paramount importance to understand the molecular mechanisms underlying the osteogenicity (osteogenic differentiation of progenitor cells in the absence of any exogenous chemical or biological osteogenic-inducing factors) and osteoinductivity (de novo bone growth in vivo even in locations where there is no vital bone) of a CaP mineral environment.The osteogenicity and osteoinductivity of CaP minerals have been attributed to different factors, such as the ability of CaP to modulate extracellular calcium (Ca²⁺) and phosphate ions and the adsorption and release of osteoinductive growth factors like bone morphogenic proteins (BMPs) (18, 21–24). This is further supported by findings that exposure of osteoblasts and progenitor cells to Ca²⁺- or -rich medium promotes their osteogenic differentiation (25–27). Additionally, it has been shown that among various CaP materials, the ones that dissociate easily to Ca²⁺ and contribute to better bone healing (13, 21). Despite the large number of studies demonstrating the potential role of CaP minerals and Ca²⁺ and on osteogenic differentiation of osteoblasts and progenitor cells, the molecular mechanism through which these ions regulate osteogenic commitment of stem cells remains largely unknown. Recent studies have shown that influx of extracellular Ca²⁺ through L-type calcium channels promotes osteogenic differentiation of osteoprogenitor cells (28). However, very little is known about the mechanism through which supports osteogenesis. During skeletal growth and bone remodeling, plays an important role in apatite formation (29, 30). In addition to osteoblasts and progenitor cells, studies have shown that exposure to alters the cell phenotype of nonskeletal tissues, such as human vascular smooth muscle cells, into osteogenic-like cells (31, 32). Central to phosphate metabolism is solute carrier family 20 (phosphate transporter), member 1 (SLC20a1, or PiT-1), a sodium-phosphate symporter that transports ions from the extracellular milieu into the cytoplasm and plays a key role in mineralization of both vascular smooth muscle cells and osteoblasts (33, 34).Here, we unravel a previously unknown mechanism, centered on phosphate metabolism, through which the CaP-rich mineral environment promotes osteogenic differentiation of human mesenchymal stem cells (hMSCs) by using an engineered matrix containing CaP moieties. Our studies show that the extracellular plays an important role in promoting osteogenic differentiation of hMSCs by regulating intramitochondrial phosphate content and ATP synthesis. ATP is then secreted and metabolized into adenosine, which promotes osteogenic differentiation of hMSCs via A2b adenosine receptors.     

Noninvasive positron emission tomography and fluorescence imaging of CD133+ tumor stem cells

A technology that visualizes tumor stem cells with clinically relevant tracers could have a broad impact on cancer diagnosis and treatment. The AC133 epitope of CD133 currently is one of the best-characterized tumor stem cell markers for many intra- and extracranial tumor entities. Here we demonstrate the successful noninvasive detection of AC133⁺ tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous and orthotopic glioma xenografts using antibody-based tracers. Particularly, microPET with ⁶⁴Cu-NOTA-AC133 mAb yielded high-quality images with outstanding tumor-to-background contrast, clearly delineating subcutaneous tumor stem cell-derived xenografts from surrounding tissues. Intracerebral tumors as small as 2–3 mm also were clearly discernible, and the microPET images reflected the invasive growth pattern of orthotopic cancer stem cell-derived tumors with low density of AC133⁺ cells. These data provide a basis for further preclinical and clinical use of the developed tracers for high-sensitivity and high-resolution monitoring of AC133⁺ tumor stem cells.Cancer stem cells (CSCs) are highly undifferentiated tumor cells with characteristics similar to normal stem cells. These characteristics include long-term replication, self-renewal, and aberrant differentiation (1, 2). Based on these characteristics, it has been hypothesized that only CSCs are able to propagate tumors for long periods of time and to initiate relapses or metastases. Furthermore, CSCs are considered to be more resistant to conventional radio- and chemotherapy than more differentiated tumor cells (3–5). Hence, elimination of CSCs is challenging but necessary for successful tumor eradication. The stem cell hypothesis of cancer development and progression is conceptually attractive and is supported by many preclinical (1, 2, 5–7) and some clinical studies (4, 8). However, larger clinical trials investigating the role of CSCs in patients have been hampered by the lack of techniques to detect, localize, and quantify the presence of CSCs noninvasively. Specifically, successful noninvasive imaging of unmanipulated CSCs with clinically relevant imaging probes (e.g., antibodies or other ligands binding CSC-specific cell-surface proteins) has not yet been reported (9–11).AC133 is an N-glycosylation–dependent epitope of the second extracellular loop of CD133/prominin-1, a cholesterol-binding protein of unknown function that locates to plasma membrane protrusions (12–14). Postnatally, the CD133 protein is expressed by certain epithelial and nonepithelial cells, by stem and progenitor cells of various organs, and by CSCs of many different types of malignant tumors (15). With a few exceptions, recognition of the AC133 epitope by the AC133 mAb appears to be limited to cells harboring stem cell properties, and the AC133 epitope—but not necessarily the CD133 protein—is down-regulated upon differentiation, presumably because of changes in glycosylation (12, 13, 15).AC133⁺ tumor stem cells have been described for glioblastoma multiforme (the most common and most aggressive primary brain tumor in adults), various pediatric brain and central nervous system tumors (medulloblastoma, ependymoma, pineoblastoma, teratoid/rhabdoid tumors, and retinoblastoma), brain metastases, many different types of carcinomas including colon, pancreatic, lung, liver, and ovarian cancer, melanoma, sarcomas, and different types of leukemia. Although AC133⁻ tumor stem cells also exist (16–18), AC133⁺ cells found in these and other tumor types have been shown to be able to self-renew, to differentiate, and to recreate the original tumors when injected into immunocompromised mice (8, 17, 19–22). Both, stemness and highly agressive malignant tumors often are associated with hypoxia (23), and hypoxia can promote the expansion of CD133⁺ cells (24). Therefore the frequent expression of AC133 on CSCs may reflect, in part, their common localization in a hypoxic environment (25).We previously reported the successful noninvasive detection of the AC133 epitope by antibody-based near-infrared fluorescence molecular tomography (NIR FMT) in mice with s.c. xenografts of CD133-overexpressing tumor cells or traditional tumor cell lines naturally displaying AC133 (26). However, we did not investigate patient-derived CSCs with the above-mentioned stem cell characteristics in that study, and NIR fluorescence, although penetrating tissues more deeply (2–4 cm) than visible-light fluorescence, has limited importance for clinical whole-body imaging (27).We report here the successful noninvasive detection of tumor-associated AC133 by PET, using a radiolabeled AC133-specific mAb in mice xenografted with tumor cell lines overexpressing CD133 or with patient-derived AC133⁺ CSCs. PET is highly sensitive and is widely used for clinical whole-body diagnostic imaging. As a PET nuclide, we used ⁶⁴Cu (t_1/2 = 12.7 h), which allows long-term tracking for at least 48 h, to follow the tumoral accumulation of relatively large molecules such as antibodies, that exhibit relatively slow tumor penetration (28). We chose S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA, hereafter abbreviated as NOTA) as the ⁶⁴Cu chelator, because high labeling efficiencies and high in vivo stability have been reported for ⁶⁴Cu-NOTA-antibody conjugates (29). In addition, we report the successful noninvasive detection of AC133⁺ CSCs with fluorescently labeled AC133 mAb and NIR imaging, a modality that is important for whole-body small-animal imaging and for intraoperative and endoscopic imaging and imaging of superficial tumors in humans (27, 30). In addition to imaging s.c. growing tumors, we report the successful antibody-mediated imaging of orthotopic xenografts initiated from AC133⁺ glioblastoma stem cells in the brain of immunocompromised mice, emphasizing the feasibility of noninvasive antibody-mediated imaging of brain tumors.     

Preferential eradication of acute myelogenous leukemia stem cells by fenretinide

Leukemia stem cells (LSCs) play important roles in leukemia initiation, progression, and relapse, and thus represent a critical target for therapeutic intervention. However, relatively few agents have been shown to target LSCs, slowing progress in the treatment of acute myelogenous leukemia (AML). Based on in vitro and in vivo evidence, we report here that fenretinide, a well-tolerated vitamin A derivative, is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells at physiologically achievable concentrations. Fenretinide exerted a selective cytotoxic effect on primary AML CD34⁺ cells, especially the LSC-enriched CD34⁺CD38⁻ subpopulation, whereas no significant effect was observed on normal counterparts. Methylcellulose colony formation assays further showed that fenretinide significantly suppressed the formation of colonies derived from AML CD34⁺ cells but not those from normal CD34⁺ cells. Moreover, fenretinide significantly reduced the in vivo engraftment of AML stem cells but not normal hematopoietic stem cells in a nonobese diabetic/SCID mouse xenotransplantation model. Mechanistic studies revealed that fenretinide-induced cell death was linked to a series of characteristic events, including the rapid generation of reactive oxygen species, induction of genes associated with stress responses and apoptosis, and repression of genes involved in NF-κB and Wnt signaling. Further bioinformatic analysis revealed that the fenretinide–down-regulated genes were significantly correlated with the existing poor-prognosis signatures in AML patients. Based on these findings, we propose that fenretinide is a potent agent that selectively targets LSCs, and may be of value in the treatment of AML.Acute myelogenous leukemia (AML) represents a group of clonal hematopoietic stem cell disorders, in which a small subpopulation of leukemia stem cells (LSCs) are responsible for the accumulation of large numbers of immature myeloblasts in the bone marrow of AML patients. In addition to their crucial roles in leukemia initiation and progression, LSCs are also responsible for the high frequency of relapse that is characteristic of current AML therapies. Of patients receiving treatment with curative intent, less than one-half will achieve long-term survival (1). Similar to normal hematopoietic stem cells (HSCs), LSCs exhibit stem cell-like characteristics such as the capacity for self-renewal, differentiation potential, and relative quiescence (2, 3). The quiescent feature renders LSCs resistant to conventional chemotherapeutic agents that predominantly target proliferating rather than quiescent cells (1). For this reason, it is not surprising that relapse occurs in the majority of cases; this is further supported by recent studies showing that AML patients with LSCs enrichment have worse clinical outcomes (4–7). It is therefore crucial that therapies be developed targeting the quiescent and drug-resistant LSCs.Despite the similarities shared by LSCs and HSCs, LSCs often possess several unique features as well, which may provide important hints for designing LSC-targeted therapy. For instance, LSCs are usually associated with the abnormal expression of CD markers (e.g., CD44, CD47, CD96, and CD123), constitutive activation of nuclear factor κB (NF-κB), active Wnt/β-catenin signaling, and elevated levels of interferon regulatory factor-1 (IRF-1) and death-associated protein kinase (DAPK) (8–12). Most recently, emerging evidence points to oxidative signaling as being a two-edged sword in AML: moderate levels of reactive oxygen species (ROS) are important for driving disease, whereas higher levels result in cell death (13–15). The dual roles of oxidative signaling suggest that LSCs, in comparison with normal HSCs, are more vulnerable to ROS-generating agents. Accordingly, pharmacological agents favoring the generation of ROS are worth exploring in LSC-targeted therapy. Indeed, ROS induction has been shown as a critical mechanism for the selective eradication of LSCs by several compounds, such as parthenolide (PTL), dimethyl-aminoparthenolide (DMAPT), 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), and 4-hydroxynonenal (HNE) (16–19).Another promising agent that could be used in this regard is fenretinide, a synthetic retinoid that lacks a carboxyl functional group likely necessary for retinoid receptor activity (20). We and others have previously demonstrated that fenretinide, unlike classical retinoids that often induce differentiation, triggers apoptotic effects; it is largely achieved through the generation of ROS (21–24), enhanced cellular ceramide, and/or ganglioside D3 (25). Moreover, several key stem cell survival-associated signaling pathways, such as NF-κB, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), have been reported to be inactivated in the fenretinide-induced apoptosis in different cancer cell types (25, 26); this further suggests the therapeutic value of fenretinide in targeting cancer stem cells.Fenretinide has been used clinically for some time as an effective chemopreventive agent for various cancers (27). It can significantly reduce the risk of breast cancer and small cell lung cancer (28, 29), suggesting an ability to prevent the development of cancer and/or eliminate early-stage malignant cells (likely cancer-initiating cells). Furthermore, long-term clinical trials have demonstrated only minimal side effects in patients receiving fenretinide (28, 30–32). In particular, no significant hematopoietic toxicity has been observed in patients treated with fenretinide (28). To illustrate the potential value of fenretinide in AML therapies, in this study, we defined the fenretinide effects on primary AML CD34⁺ cells and LSC-enriched AML CD34⁺CD38⁻ cells.     

Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling

We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.Large-scale sequencing analyses of solid cancers have identified extensive tumor heterogeneity within individual primary cancers (1). Recent studies indicate that such tumoral heterogeneity is associated with heterogeneous protein function, which fosters tumor adaptation, treatment resistance, and failure through Darwinian selection (2–4). Cancer stem cells are a subpopulation of cells within the primary tumor responsible for tumor initiation and metastases (5–9). Three groups have recently independently provided functional evidence for the presence of cancer stem cells by lineage-tracing experiments (10–12). These observations suggest that these subpopulations of cancer stem cells (CSCs) within the bulk primary tumor are resistant to conventional therapies through different adaptive mechanisms with the potential for self-renewal and metastases (7, 13, 14). However, few studies have determined the genetic profile of the cells that escape the primary cancer and evolve in distant metastatic sites (1). Additionally, no large-scale sequencing studies of metastases have been conducted because the majority of patients are treated with systemic therapies and not surgery.Tumor clonal heterogeneity within a primary tumor may in part be explained by hypoxic regions within the bulk tumor that have been correlated with invasiveness, therapeutic resistance, and metastasis (15–18). Cancer stem cells have been found to reside near hypoxic regions in some solid cancers (19–21). We have previously published a 477-gene tumorigenic signature by isolating breast cancer stem cells (BCSCs) derived from patient biopsies (22). Here, we have identified two previously unidentified cancer genes, ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2), by selective shRNA knockdown of genes from this tumorigenic signature, that impact breast cancer stem cell self-renewal and lung metastases. Analysis of 53 patient lung metastases confirmed damaging mutations in RPL39 and MLF2 in a significant number of samples, which conferred a gain-of-function phenotype. These mutations were statistically associated with shorter median time to distant relapse. We further describe a common mechanism of action through nitric oxide synthase signaling that is regulated by hypoxia.     

CD14-expressing cancer cells establish the inflammatory and proliferative tumor microenvironment in bladder cancer

Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.Solid tumors represent a complex mass of tissue composed of multiple distinct cell types (1, 2). Cells within the tumor produce a range of soluble factors to create a complex of signaling networks within the tumor microenvironment (3–7). One of the outcomes of this crosstalk is tumor-promoting inflammation (TPI) (8, 9). TPI can modulate the functions of tumor-infiltrating myeloid lineage cells including macrophages (10–12). Tumor-associated macrophages (TAMs) consistently display an alternatively activated phenotype (M2) commonly found in sites of wound healing (13–18). These macrophages promote tumor growth while suppressing the host immune response locally (19–22). Polarization and subversion of tumor-infiltrating macrophages is accomplished via immune mediators in the tumor microenvironment (23, 24). Adding to the complexity of solid tumors is the heterogeneity of the cancer cells (2). Tumor cells of varying differentiation states and different characteristics coexist within a tumor (25–29). However, the different roles of each tumor cell subset during cancer progression remain undefined.Bladder cancer (BC) represents a growing number of solid tumors characterized by the infiltration of a significant number of myeloid cells in the neoplastic lesion (30, 31). We have previously determined that keratin 14 (KRT14) expression marks the most primitive differentiation state in BC cells (32). KRT14 expression is significantly associated with poor overall patient survival. However, the mechanisms used by KRT14-expressing cells to promote tumor growth remain unclear. In the current study, we found that KRT14+ basal BC cells also express higher levels of CD14. Here, we investigate the strategies used by KRT14+ CD14-high BC cells to promote tumor growth.     

Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles

Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.Mammalian females ovulate periodically over their reproductive lifetimes, placing significant demands on their ovaries for oocyte production. Murine females produce up to 500 oocytes during 50 cycles, whereas human females release a similar number during 40 y of monthly cycles. Before birth, their ovaries contain thousands (mice) or millions (human) of prefollicular germ cells. A large “ovarian reserve” of primordial follicles is generated around the time of birth from prefollicular germ cells, and follicle numbers decline slowly (1). Because histological evidence of prefollicular germ cells also disappears at birth, it has been widely thought that the initial follicles are stable enough to sustain oogenesis throughout the normal reproductive lifespan (2).During the last decade, it has been claimed that primordial follicles in adult ovaries are highly unstable and that mouse and human females consequently require adult germ-line stem cells (GSCs) to maintain a pool of follicles and sustain ovulation. Active stem cells were placed in the ovarian surface epithelium (3) or in the bone marrow (4). However, others failed to reproduce these data and their predictions (5–10). Subsequently, evidence for ovarian GSCs came from transplantation assays. Selected ovarian cells explanted into culture gave rise to rare cells capable of forming oocytes following transplantation into a host ovary (11, 12). This work has also been challenged (13). Recently, patterns of somatic mutations in female germ cells during adulthood were interpreted to be consistent with the presence of adult stem cells (14). Normal adult female GSCs, should they exist, might prove useful for advancing reproductive health and for stem-cell-based therapies. Lineage tracing in vivo constitutes the definitive method for detecting stem cells (15). Here we show by single-cell lineage tracing that GSCs are not required to maintain the mouse primordial follicle pool and are undetectable in adult mouse ovaries.     

Drug-eluting microarrays to identify effective chemotherapeutic combinations targeting patient-derived cancer stem cells

A new paradigm in oncology establishes a spectrum of tumorigenic potential across the heterogeneous phenotypes within a tumor. The cancer stem cell hypothesis postulates that a minute fraction of cells within a tumor, termed cancer stem cells (CSCs), have a tumor-initiating capacity that propels tumor growth. An application of this discovery is to target this critical cell population using chemotherapy; however, the process of isolating these cells is arduous, and the rarity of CSCs makes it difficult to test potential drug candidates in a robust fashion, particularly for individual patients. To address the challenge of screening drug libraries on patient-derived populations of rare cells, such as CSCs, we have developed a drug-eluting microarray, a miniaturized platform onto which a minimal quantity of cells can adhere and be exposed to unique treatment conditions. Hundreds of drug-loaded polymer islands acting as drug depots colocalized with adherent cells are surrounded by a nonfouling background, creating isolated culture environments on a solid substrate. Significant results can be obtained by testing <6% of the cells required for a typical 96-well plate. Reliability was demonstrated by an average coefficient of variation of 14% between all of the microarrays and 13% between identical conditions within a single microarray. Using the drug-eluting array, colorectal CSCs isolated from two patients exhibited unique responses to drug combinations when cultured on the drug-eluting microarray, highlighting the potential as a prognostic tool to identify personalized chemotherapeutic regimens targeting CSCs.Tumor-initiating cancer stem cells (CSCs) are being investigated as a promising therapeutic target (1). The rarity of CSCs, which constitute ∼1% of tumor cells (1, 2), limits their availability for testing, and traditional screening methods require substantial cell quantities. Industrial pharmaceutical capabilities have successfully reduced cell requirements in drug screening, but such capital-intensive facilities are typically unavailable to clinicians and pathology laboratories. The past decade has witnessed the emergence of multiple cell-based microarray platforms that address availability and cell source limitations (3–5), although these systems have inherent shortcomings. Many rely on immobilizing target molecules (6–9), limiting applicability to small molecule drug libraries, whereas others rely on robotically spotting cells (10), a technique not amenable to widespread adoption. Array platforms capable of capturing single cells have been established (11, 12), but determination of chemotherapeutic efficacy is better investigated through methods using greater cell numbers, which better capture variability in cellular responses. Furthermore, arrays of drug-loaded polymer films with an overlying cell monolayer have been developed (13), but monolayers of cells are susceptible to juxtacrine and paracrine signaling, which are particularly important for multipotent cells. In the present work, the provision of differential cell adhesion to promote seeding onto spotted drug-loaded films against a surrounding nonfouling background (i.e., a surface that resists protein adsorption and thus cell adhesion) can separate drug-eluting polymer films to create isolated culture environments. The use of programmable arraying techniques can then enable fabrication of uniquely formulated drug-eluting spots that provide prescribed drug doses and drug combinations to overlying cells for simultaneous testing on a single device.It is becoming increasingly evident in cancer treatment that simultaneously targeting multiple critical pathways using combinations of chemotherapeutic drugs can enhance outcomes (14–17). Conventional screening of chemotherapeutics uses an established panel of cancer cell lines (18) that have been derived from bulk tumors. A recently developed clinical approach involves performing in vitro chemosensitivity testing of tumor biopsy specimens to individualize treatment (19, 20). Unfortunately, benefits have been limited, with poor correlations between bulk tumor cell sensitivity and clinical efficacy. This lack of efficacy has been attributed to patient to patient variability, owing in part to intratumor heterogeneity (21–23).Tumors consist of multiple cell phenotypes. In the CSC model, a rare cell population of tumor-initiating cells perpetually self-renew and are responsible for tumor heterogeneity, metastasis, and disease recurrence (1, 24). Recent identification of unique cell surface markers that enrich tumor cell isolates for CSCs have led to novel techniques for isolating enriched colorectal CSC (CCSC) populations from patient tumor samples (25–28). For example, xenotransplantation of a single CCSC identified by high Wnt/Β-catenin signaling activity generates tumors that recapitulate the diverse phenotypic heterogeneity of the original tumor (29). Thus, identifying and isolating CCSCs out of the tumor bulk from an individual cancer patient and determining sensitivity to chemotherapeutic drugs in vitro is possible (30, 31). An approach such as the drug-eluting microarray, enabling use of low cell numbers, could potentiate personalized combination drug treatment screens for efficacy against patient-specific CSCs.