Dynamic Control Strategy to Produce Riboflavin with Lignocellulose Hydrolysate in the Thermophile Geobacillus thermoglucosidasius.

Efficient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here, we report a GCR-based dynamic control (GCR-DC) strategy aiming at better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of riboflavin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM 2542 as a host, we constructed additional riboflavin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the first stage, glucose was preferentially used for cell growth and no production of riboflavin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for riboflavin biosynthesis. Using corn cob hydrolysate as a carbon source, the optimized riboflavin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single-module dynamic control strategy) were 5.3- and 2.3-fold higher than that of the control strain DSM 2542 Rib-Gtg constitutively producing riboflavin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can efficiently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.

Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus.

Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications. The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (ΔpyrF) resistant to the highly toxic uracil analog of 5-fluoroorotic acid (5-FOA) converted by PyrF, and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase. The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications. Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutant ΔpyrF at the targeted locus. Double-crossover recombinants were generated, from which the pyrF gene, plasmid backbone, and targeted gene were excised through homologous recombination exchange. These recombinants were rapidly screened by the counterselection agent, 5-FOA. We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018. This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.

Comparison of postoperative analgesic effects in response to either dexamethasone or dexmedetomidine as local anesthetic adjuvants: a systematic review and meta-analysis of randomized controlled trials.

This review compares the effects of peripheral dexamethasone and dexmedetomidine on postoperative analgesia. We included six randomized controlled trials (354 patients) through a systematic literature search. We found that analgesia duration was comparable between dexamethasone and dexmedetomidine (58.59 min, 95% CI (confidence interval), - 66.13, 183.31 min) with extreme heterogeneity. Secondary outcome was also compared and no significant difference was observed in sensory block onset and duration and motor block duration and also for postoperative nausea and vomiting. It is noteworthy that dexamethasone reduced analgesic consumption (fentanyl) by 29.12 mcg compared with dexmedetomidine. We performed subgroup analyses and found no significant difference between the following: (1) lidocaine vs ropivacaine (P = 0.28), (2) nerve block vs nerve block + general anesthesia (P = 0.47), and (3) upper limb surgery vs thoracoscopic pneumonectomy (P = 0.27). We applied trial sequential analysis to assess the risks of type I and II errors and concluded that the meta-analysis was insufficiently powered to answer the clinical question, and further analysis is needed to establish which adjuvant is better. In conclusion, we believe that existing research indicates that dexamethasone and dexmedetomidine have equivalent analgesic effects in peripheral nerve blocks.

Coordinating precursor supply for pharmaceutical polyketide production in Streptomyces.

The widely used polyketide pharmaceuticals in medicine and agriculture are mainly produced by Streptomyces species. These compounds, as secondary metabolites, are not involved in essential cellular processes and are usually produced during the stationary phase of fermentation. Consequently, their yields and productivities are often low and frequently limited by the availability of the precursors. The precursor pathways, therefore, are key entities for synthetic biology-driven design and optimization. We discuss recent advances in precursor engineering, in both Streptomyces and other bacteria, focusing on the diverse native and heterologous precursor pathways that could be rewired for polyketide titer improvement. We also highlight the coordination of other required factors to direct the precursors towards polyketide biosynthesis. The precursor-supply enhancement tools and strategies covered in this review will facilitate the design and construction of synthetic Streptomyces 'cell-factories' for efficient polyketide production.

An Antioxidant Enzyme Therapeutic for COVID-19.

The COVID-19 pandemic has taken a significant toll on people worldwide, and there are currently no specific antivirus drugs or vaccines. Herein it is a therapeutic based on catalase, an antioxidant enzyme that can effectively breakdown hydrogen peroxide and minimize the downstream reactive oxygen species, which are excessively produced resulting from the infection and inflammatory process, is reported. Catalase assists to regulate production of cytokines, protect oxidative injury, and repress replication of SARS-CoV-2, as demonstrated in human leukocytes and alveolar epithelial cells, and rhesus macaques, without noticeable toxicity. Such a therapeutic can be readily manufactured at low cost as a potential treatment for COVID-19.

Age dependent effects of NELL-1 isoforms on bone marrow stromal cells.

NELL-1 is an osteogenic protein first discovered to control ossification of the cranium. NELL-1 exists in at least two isoforms. The full-length NELL-1 contains 810 amino acid (aa) (NELL-1810), the N-terminal-truncated NELL-1 isoform contains 570 aa (NELL-1570). The differences in cellular effects between NELL-1 isoforms are not well understood. Methods: Here, BMSC were derived from adult or aged mice, followed by overexpression of NELL-1810 or NELL-1570. Cell morphology, proliferation, and gene expression were examined. Results/Conclusions: Overall, the proliferative effect of NELL-1570 was age dependent, showing prominent induction in adult but not aged mice.

Peroxisome Proliferator-Activated Receptor-γ Knockdown Impairs Bone Morphogenetic Protein-2-Induced Critical-Size Bone Defect Repair.

The Food and Drug Administration-approved clinical dose (1.5 mg/mL) of bone morphogenetic protein-2 (BMP2) has been reported to induce significant adverse effects, including cyst-like adipose-infiltrated abnormal bone formation. These undesirable complications occur because of increased adipogenesis, at the expense of osteogenesis, through BMP2-mediated increases in the master regulatory gene for adipogenesis, peroxisome proliferator-activated receptor-γ (PPARγ). Inhibiting PPARγ during osteogenesis has been suggested to drive the differentiation of bone marrow stromal/stem cells toward an osteogenic, rather than an adipogenic, lineage. We demonstrate that knocking down PPARγ while concurrently administering BMP2 can reduce adipogenesis, but we found that it also impairs BMP2-induced osteogenesis and leads to bone nonunion in a mouse femoral segmental defect model. In addition, in vitro studies using the mouse bone marrow stromal cell line M2-10B4 and mouse primary bone marrow stromal cells confirmed that PPARγ knockdown inhibits BMP2-induced adipogenesis; attenuates BMP2-induced cell proliferation, migration, invasion, and osteogenesis; and escalates BMP2-induced cell apoptosis. More important, BMP receptor 2 and 1B expression was also significantly inhibited by the combined BMP2 and PPARγ knockdown treatment. These findings indicate that PPARγ is critical for BMP2-mediated osteogenesis during bone repair. Thus, uncoupling BMP2-mediated osteogenesis and adipogenesis using PPARγ inhibition to combat BMP2's adverse effects may not be feasible.

Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis.

Contactin-associated protein-like 4 (Cntnap4) is a member of the neurexin superfamily of transmembrane molecules that have critical functions in neuronal cell communication. Cntnap4 knockout mice display decreased presynaptic gamma-aminobutyric acid (GABA) and increased dopamine release that is associated with severe, highly penetrant, repetitive, and perseverative movements commonly found in human autism spectrum disorder patients. However, no known function of Cntnap4 has been revealed besides the nervous system. Meanwhile, secretory protein neural EGFL-like 1 (Nell-1) is known to exert potent osteogenic effects in multiple small and large animal models without the off-target effects commonly found with bone morphogenetic protein 2. In this study, while searching for a Nell-1-specific cell surface receptor during osteogenesis, we identified and validated a ligand/receptor-like interaction between Nell-1 and Cntnap4 by demonstrating: 1) Nell-1 and Cntnap4 colocalization on the surface of osteogenic-committed cells; 2) high-affinity interaction between Nell-1 and Cntnap4; 3) abrogation of Nell-1-responsive Wnt and MAPK signaling transduction, as well as osteogenic effects, via Cntnap4 knockdown; and 4) replication of calvarial cleidocranial dysplasias-like defects observed in Nell-1-deficient mice in Wnt1-Cre-mediated Cntnap4-knockout transgenic mice. In aggregate, these findings indicate that Cntnap4 plays a critical role in Nell-1-responsive osteogenesis. Further, this is the first functional annotation for Cntnap4 in the musculoskeletal system. Intriguingly, Nell-1 and Cntnap4 also colocalize on the surface of human hippocampal interneurons, implicating Nell-1 as a potential novel ligand for Cntnap4 in the nervous system. This unexpected characterization of the ligand/receptor-like interaction between Nell-1 and Cntnap4 indicates a novel biological functional axis for Nell-1 and Cntnap4 in osteogenesis and, potentially, in neural development and function. © 2018 American Society for Bone and Mineral Research.

Frontal Bone Healing Is Sensitive to Wnt Signaling Inhibition via Lentiviral-Encoded Beta-Catenin Short Hairpin RNA.

The Wnt/ß-catenin signaling pathway plays an integral role in skeletal biology, spanning from embryonic skeletal patterning through bone maintenance and bone repair. Most experimental methods to antagonize Wnt signaling in vivo are either systemic or transient, including genetic approaches, use of small-molecule inhibitors, or neutralizing antibodies. We sought to develop a novel, localized model of prolonged Wnt/ß-catenin signaling blockade by the application and validation of a lentivirus encoding ß-catenin short hairpin RNA (shRNA). Efficacy of lentiviral-encoded ß-catenin shRNA was first confirmed in vitro using bone marrow mesenchymal stromal cells, and in vivo using an intramedullary long bone injection model in NOD SCID mice. Next, the effects of ß-catenin knockdown were assessed in a calvarial bone defect model, in which the frontal bone demonstrates enhanced bone healing associated with heightened Wnt/ß-catenin signaling. Lentivirus encoding either ß-catenin shRNA or random sequence shRNA with enhanced green fluorescent protein (control) was injected overlying the calvaria of NOD SCID mice and bone defects were created in either the frontal or parietal bones. Among mice treated with lentivirus encoding ß-catenin shRNA, frontal bone defect healing was significantly reduced by all radiographic and histologic metrics. In contrast, parietal bone healing was minimally impacted by ß-catenin shRNA. In aggregate, our data document the application and validation of a lentivirus encoding ß-catenin shRNA model that represents an easily replicable tool for examining the importance of locoregional Wnt/ß-catenin signaling in bone biology and regeneration.

Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2.

The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis.

Discovery and validation of potential bacterial biomarkers for lung cancer.

Microbes are residents in a number of body sites, including the oral and nasal cavities, which are connected to the lung via the pharynx. The associations between oral diseases and increased risk of lung cancer have been reported in previous prospective studies. In this study, we measured variations of salivary microbiota and evaluated their potential association with lung cancer, including squamous cell carcinoma (SCC) and adenocarcinoma (AC). A three-phase study was performed: First, we investigated the salivary microbiota from 20 lung cancer patients (10 SCC and 10 AC) and control subjects (n=10) using a deep sequencing analysis. Salivary Capnocytophaga, Selenomonas, Veillonella and Neisseria were found to be significantly altered in patients with SCC and AC when compared to that in control subjects. Second, we confirmed the significant changes of Capnocytophaga, Veillonella and Neisseria in the same lung cancer patients using quantitative PCR (qPCR). Finally, these bacterial species were further validated on new patient/control cohorts (n=56) with qPCR. The combination of two bacterial biomarkers, Capnocytophaga and Veillonella, yielded a receiver operating characteristic (ROC) value of 0.86 with an 84.6% sensitivity and 86.7% specificity in distinguishing patients with SCC from control subjects and a ROC value of 0.80 with a 78.6% sensitivity and 80.0% specificity in distinguishing patients with AC from control subjects. In conclusion, we have for the first time demonstrated the association of saliva microbiota with lung cancer. Particularly, the combination of the 16S sequencing discovery with qPCR validation studies revealed that the levels of Capnocytophaga and Veillonella were significantly higher in the saliva from lung cancer patients, which may serve as potential biomarkers for the disease detection/classification.

Proliferation and osteogenic differentiation of mesenchymal stem cells induced by a short isoform of NELL-1.

Neural epidermal growth factor-like (NEL)-like protein 1 (NELL-1) has been identified as an osteoinductive differentiation factor that promotes mesenchymal stem cell (MSC) osteogenic differentiation. In addition to full-length NELL-1, there are several NELL-1-related transcripts reported. We used rapid amplification of cDNA ends to recover potential cDNA of NELL-1 isoforms. A NELL-1 isoform with the N-terminal 240 amino acid (aa) residues truncated was identified. While full-length NELL-1 that contains 810 aa residues (NELL-1810 ) plays an important role in embryologic skeletal development, the N-terminal-truncated NELL-1 isoform (NELL-1570 ) was expressed postnatally. Similar to NELL-1810 , NELL-1570 induced MSC osteogenic differentiation. In addition, NELL-1570 significantly stimulated MSC proliferation in multiple MSC-like populations such as murine C3H10T1/2 MSC cell line, mouse primary MSCs, and perivascular stem cells, which is a type of stem cells proposed as the perivascular origin of MSCs. In contrast, NELL-1810 demonstrated only limited stimulation of MSC proliferation. Similar to NELL-1810 , NELL-1570 was found to be secreted from host cells. Both NELL-1570 expression lentiviral vector and column-purified recombinant protein NELL-1570 demonstrated almost identical effects in MSC proliferation and osteogenic differentiation, suggesting that NELL-1570 may function as a pro-osteogenic growth factor. In vivo, NELL-1570 induced significant calvarial defect regeneration accompanied by increased cell proliferation. Thus, NELL-1570 has the potential to be used for cell-based or hormone-based therapy of bone regeneration.

High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA.

The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences) of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

Lentiviral delivery of PPARγ shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture.

INTRODUCTION: Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPARγ) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPARγ shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model. METHODS: PPARγ shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPARγ shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPARγ, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K). RESULTS: In vitro, PPARγ shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPARγ shRNA treatment. DISCUSSION: These findings suggest that acute loss of PPARγ in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPARγ shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPARγ shRNA on OB and OC cell types, and it may help determine whether PPARγ antagonists are potential therapeutic agents for osteoporotic bone loss.

Perivascular stem cells: a prospectively purified mesenchymal stem cell population for bone tissue engineering.

Adipose tissue is an ideal source of mesenchymal stem cells for bone tissue engineering: it is largely dispensable and readily accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which leads to unreliable bone formation. In the present study, we prospectively purified human perivascular stem cells (PSCs) from adipose tissue and compared their bone-forming capacity with that of traditionally derived SVF. PSCs are a population (sorted by fluorescence-activated cell sorting) of pericytes (CD146+CD34-CD45-) and adventitial cells (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. Here, we found that PSCs underwent osteogenic differentiation in vitro and formed bone after intramuscular implantation without the need for predifferentiation. We next sought to optimize PSCs for in vivo bone formation, adopting a demineralized bone matrix for osteoinduction and tricalcium phosphate particle formulation for protein release. Patient-matched, purified PSCs formed significantly more bone in comparison with traditionally derived SVF by all parameters. Recombinant bone morphogenetic protein 2 increased in vivo bone formation but with a massive adipogenic response. In contrast, recombinant Nel-like molecule 1 (NELL-1; a novel osteoinductive growth factor) selectively enhanced bone formation. These studies suggest that adipose-derived human PSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, PSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy. Finally, NELL-1 is a candidate growth factor able to induce human PSC osteogenesis.

An abundant perivascular source of stem cells for bone tissue engineering.

Adipose tissue is an ideal mesenchymal stem cell (MSC) source, as it is dispensable and accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which has disadvantages for tissue regeneration. In the present study, we prospectively purified human perivascular stem cells (PSCs) from n = 60 samples of human lipoaspirate and documented their frequency, viability, and variation with patient demographics. PSCs are a fluorescence-activated cell sorting-sorted population composed of pericytes (CD45-, CD146+, CD34-) and adventitial cells (CD45-, CD146-, CD34+), each of which we have previously reported to have properties of MSCs. Here, we found that PSCs make up, on average, 43.2% of SVF from human lipoaspirate (19.5% pericytes and 23.8% adventitial cells). These numbers were minimally changed by age, gender, or body mass index of the patient or by length of refrigerated storage time between liposuction and processing. In a previous publication, we observed that human PSCs (hPSCs) formed significantly more bone in vivo in comparison with unsorted human SVF (hSVF) in an intramuscular implantation model. We now extend this finding to a bone injury model, observing that purified hPSCs led to significantly greater healing of mouse critical-size calvarial defects than hSVF (60.9% healing as opposed to 15.4% healing at 2 weeks postoperative by microcomputed tomography analysis). These studies suggest that adipose-derived hPSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, hPSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy.

Involvement of non-structural proteins (NS) in influenza A infection and viral tropism.

Hemagglutinin (HA) of influenza A has been reported as the key protein in viral infection. Therefore, the density and the dynamic pattern of this protein in viral envelope will affect the virus to infect target cells. We used a lentiviral system to study the influenza A H1N1 viral infection. Herein we demonstrate that the influenza non-structural proteins (NS) significantly promote viral infection. By substituting NS gene segment from an H1N1 genome set of A/WSN/1933 with the NS segment isolated from another H1N1 substrain genome set, China246, we found that viral infection tropism was significantly altered. The reassortant H1N1 shows almost identical infectivity compared with its parental virus, A/WSN/1933, for the human epithelial cell line HOT, but shows only 1/100 infectivity of its parental virus when infecting the Madin-Darby canine kidney (MDCK) cell line. These results suggest that not only is NS important in the infectivity of human influenza virus, but that it may play a critical role in viral tropism, allowing the virus to mutate and spread to other species.

Reprogramming of human fibroblasts into multipotent cells with a single ECM proteoglycan, fibromodulin.

Pluripotent and/or multipotent stem cell-based therapeutics are a vital component of tissue engineering and regenerative medicine. The generation or isolation of safer and readily available stem cell sources will significantly aid clinical applications. We report here a technique using a single molecule, recombinant human fibromodulin protein (FMOD), to reprogram human fibroblasts into multipotent cells. Like virally-induced pluripotent stem (iPS) cells, FMOD reprogrammed (FReP) cells express pluripotency markers, form embryoid bodies (EBs), and differentiate into ectoderm, mesoderm, and endoderm derivatives in vitro. Notably, FReP cells regenerate muscle and bone tissues but do not generate teratomas in vivo. Unlike iPS cells, undifferentiated FReP cells proliferate slowly and express low proto-oncogene c-MYC and unexpectedly high levels of cyclin-dependent kinase inhibitors p15(Ink4B) and p21(WAF1/Cip1). Remarkably, in a fashion reminiscent of quiescent stem cells, the slow replicative phenotype of undifferentiated FReP cells reverses after differentiation induction, with differentiating FReP cells proliferating faster and expressing less p15(Ink4B) and p21(WAF1/Cip1) than differentiating iPS cells. Overall, single protein, FMOD-based, cell reprograming bypasses the risks of mutation, gene instability, and malignancy associated with genetically-modified iPS cells, and provides an alternative strategy for engineering patient-specific multipotent cells for basic research and therapeutic application.

The PSA(-/lo) prostate cancer cell population harbors self-renewing long-term tumor-propagating cells that resist castration.

Prostate cancer (PCa) is heterogeneous and contains both differentiated and undifferentiated tumor cells, but the relative functional contribution of these two cell populations remains unclear. Here we report distinct molecular, cellular, and tumor-propagating properties of PCa cells that express high (PSA(+)) and low (PSA(-/lo)) levels of the differentiation marker PSA. PSA(-/lo) PCa cells are quiescent and refractory to stresses including androgen deprivation, exhibit high clonogenic potential, and possess long-term tumor-propagating capacity. They preferentially express stem cell genes and can undergo asymmetric cell division to generate PSA(+) cells. Importantly, PSA(-/lo) PCa cells can initiate robust tumor development and resist androgen ablation in castrated hosts, and they harbor highly tumorigenic castration-resistant PCa cells that can be prospectively enriched using ALDH(+)CD44(+)α2ß1(+) phenotype. In contrast, PSA(+) PCa cells possess more limited tumor-propagating capacity, undergo symmetric division, and are sensitive to castration. Altogether, our study suggests that PSA(-/lo) cells may represent a critical source of castration-resistant PCa cells.