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Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium,functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts,we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system,these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs,we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust,scalable,and consistent methodology. In the present study,we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set,we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality,with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed,demonstrating the value of this biologically relevant and reproducible technology. In addition,this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells. View Publication

Premature proliferative arrest in benign or early-stage tumors induced by oncoproteins,chromosomal instability,or DNA damage is associated with p53/p21 activation,culminating in either senescence or apoptosis,depending on cell context. Growth hormone (GH) elicits direct peripheral metabolic actions as well as growth effects mediated by insulin-like growth factor 1 (IGF1). Locally produced peripheral tissue GH,in contrast to circulating pituitary-derived endocrine GH,has been proposed to be both proapoptotic and prooncogenic. Pituitary adenomas expressing and secreting GH are invariably benign and exhibit DNA damage and a senescent phenotype. We therefore tested effects of nutlin-induced p53-mediated senescence in rat and human pituitary cells. We show that DNA damage senescence induced by nutlin triggers the p53/p21 senescent pathway,with subsequent marked induction of intracellular pituitary GH in vitro. In contrast,GH is not induced in cells devoid of p53. Furthermore we show that p53 binds specific GH promoter motifs and enhances GH transcription and secretion in senescent pituitary adenoma cells and also in nonpituitary (human breast and colon) cells. In vivo,treatment with nutlin results in up-regulation of both p53 and GH in the pituitary gland,as well as increased GH expression in nonpituitary tissues (lung and liver). Intracrine GH acts in pituitary cells as an apoptosis switch for p53-mediated senescence,likely protecting the pituitary adenoma from progression to malignancy. Unlike in the pituitary,in nonpituitary cells GH exerts antiapoptotic properties. Thus,the results show that GH is a direct p53 transcriptional target and fulfills criteria as a p53 target gene. Induced GH is a readily measurable cell marker for p53-mediated cellular senescence. View Publication

INTRODUCTION Hirschsprung's disease is characterized by a developmental arrest of neural crest cell migration,causing distal aganglionosis. Transplanted cells derived from the neural crest may regenerate enteric ganglia in this condition. We investigated the potential of skin-derived precursor cells (SKPs) to engraft and to differentiate into enteric ganglia in aganglionic rat intestine in vivo. METHODS Adult Lewis rat jejunal segments were separated from intestinal continuity and treated with benzalkonium chloride to induce aganglionosis. Ganglia were identified via immunohistochemical stains for S100 and β-III tubulin (TUJ1). SKPs were procured from neonatal Lewis rats expressing enhanced green fluorescent protein (GFP) and cultured in neuroglial-selective media. SKP cell line expansion was quantified,and immunophenotypes were assessed by immunocytochemistry. Aganglionic segments underwent SKP transplantation 21-79days after benzalkonium chloride treatment. The presence of GFP+cells,mature neurons,and mature glia was evaluated at posttransplant days 1,6,and 9. RESULTS Benzalkonium chloride-induced aganglionosis persisted for at least 85days. Prior to differentiation,SKPs expressed S100,denoting neural crest lineage,and nestin,a marker of neuronal precursors. Differentiated SKPs in vitro expressed GFAP,a marker of glial differentiation,as well as TUJ1 and several enteric neurotransmitters. After transplantation,GFP+structures resembling ganglia were identified between longitudinal and circular smooth muscle layers. CONCLUSION SKPs are capable of engraftment,migration,and differentiation within aganglionic rodent intestine in vivo. Differentiated SKPs generate structures that resemble enteric ganglia. Our observations suggest that SKPs represent a potential gangliogenic therapeutic agent for Hirschsprung's disease. View Publication

Chimeric antigen receptor (CAR) therapy targeting CD19 has yielded remarkable outcomes in patients with acute lymphoblastic leukemia. To identify potential CAR targets in acute myeloid leukemia (AML),we probed the AML surfaceome for overexpressed molecules with tolerable systemic expression. We integrated large transcriptomics and proteomics datasets from malignant and normal tissues,and developed an algorithm to identify potential targets expressed in leukemia stem cells,but not in normal CD34+CD38- hematopoietic cells,T cells,or vital tissues. As these investigations did not uncover candidate targets with a profile as favorable as CD19,we developed a generalizable combinatorial targeting strategy fulfilling stringent efficacy and safety criteria. Our findings indicate that several target pairings hold great promise for CAR therapy of AML. View Publication

The generation of differentiated and functional neurons is a complex process,which requires coordinated expression of several proteins and microRNAs (miRNAs). The present study using nerve growth factor (NGF)-differentiated PC12 cells led to the identification of miR-200,miR-221/222 and miR-34 families as major up-regulated miRNAs in fully differentiated neurons. Similar to PC12 cells,induction of miR-200 family was observed in differentiating neural stem cells,demonstrating a direct role of miR-200 family in neuronal differentiation. Over-expression of miR-200 induced neurite formation in PC12 cells and regulated neuronal markers in favour of differentiation. However,inhibition of miR-200 induced proliferation of PC12 cells. In differentiating PC12 cells and neural stem cells,an inverse relationship was observed between expression of reprogramming transcription factors (SOX2,KLF4,NANOG,OCT4 and PAX6) and miR-200. Over-expression of miR-200 in PC12 cells significantly down-regulated mRNA and protein levels of SOX2 and KLF4. Moreover,we observed two phases of dramatic down-regulation of miR-200 expression in developing rat brains correlating with periods of neuronal proliferation. In conclusion,our results indicate that increased expression of the miR-200 family promotes neuronal differentiation,while decreased expression of the miR-200 family promotes neuronal proliferation by targeting SOX2 and KLF4. View Publication

Recently there has been growing interest in the differentiation of mesenchymal stem cells (MSCs) into neural lineages. Research suggests that MSCs can be differentiated into neural progenitor-like cells (NPCs) under the specific influence of paracrine factors particularly epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Our recent research has found that the addition of insulin-like growth factor 1 (IGF-1) with the combination of the EGF and bFGF could significantly improve the growth and survivability of MSC-derived NPCs. To unravel the molecular mechanism of the improved differentiation we compared the microRNA expression profiles of the differentiation under various combinations of growth factors. MSCs were differentiated into neural lineage in 3 groups; Group A (EGF + bFGF),Group B (EGF + bFGF + IGF-1),and Group C (without growth factor). Regulated microRNAs during the early differentiation were identified by detailed microRNA profiling using Affymetrix GeneChip version 2.0 at three time intervals (day 1,day 3 and day 5). The data were deposited in the Gene Expression Omnibus,series GSE60060. View Publication

BACKGROUND There has been increasing interest recently in the plasticity of mesenchymal stem cells (MSCs) and their potential to differentiate into neural lineages. To unravel the roles and effects of different growth factors in the differentiation of MSCs into neural lineages,we have differentiated MSCs into neural lineages using different combinations of growth factors. Based on previous studies of the roles of insulin-like growth factor 1 (IGF-1) in neural stem cell isolation in the laboratory,we hypothesized that IGF-1 can enhance proliferation and reduce apoptosis in neural progenitor-like cells (NPCs) during differentiation of MSCs into NCPs.We induced MSCs differentiation under four different combinations of growth factors: (A) EGF%+%bFGF,(B) EGF%+%bFGF%+%IGF-1,(C) EGF%+%bFGF%+%LIF,(D) EGF%+%bFGF%+%BDNF,and (E) without growth factors,as a negative control. The neurospheres formed were characterized by immunofluorescence staining against nestin,and the expression was measured by flow cytometry. Cell proliferation and apoptosis were also studied by MTS and Annexin V assay,respectively,at three different time intervals (24 hr,3 days,and 5 days). The neurospheres formed in the four groups were then terminally differentiated into neuron and glial cells. RESULTS The four derived NPCs showed a significantly higher expression of nestin than was shown by the negative control. Among the groups treated with growth factors,NPCs treated with IGF-1 showed the highest expression of nestin. Furthermore,NPCs derived using IGF-1 exhibited the highest cell proliferation and cell survival among the treated groups. The NPCs derived from IGF-1 treatment also resulted in a better yield after the terminal differentiation into neurons and glial cells than that of the other treated groups. CONCLUSIONS Our results suggested that IGF-1 has a crucial role in the differentiation of MSCs into neuronal lineage by enhancing the proliferation and reducing the apoptosis in the NPCs. This information will be beneficial in the long run for improving both cell-based and cell-free therapy for neurodegenerative diseases. View Publication

Application of high-content immune profiling technologies has enormous potential to advance medicine. Whether these technologies reveal pertinent biology when implemented in interventional clinical trials is an important question. The beneficial effects of preoperative arginine-enriched dietary supplements (AES) are highly context specific,as they reduce infection rates in elective surgery,but possibly increase morbidity in critically ill patients. This study combined single-cell mass cytometry with the multiplex analysis of relevant plasma cytokines to comprehensively profile the immune-modifying effects of this much-debated intervention in patients undergoing surgery. An elastic net algorithm applied to the high-dimensional mass cytometry dataset identified a cross-validated model consisting of 20 interrelated immune features that separated patients assigned to AES from controls. The model revealed wide-ranging effects of AES on innate and adaptive immune compartments. Notably,AES increased STAT1 and STAT3 signaling responses in lymphoid cell subsets after surgery,consistent with enhanced adaptive mechanisms that may protect against postsurgical infection. Unexpectedly,AES also increased ERK and P38 MAPK signaling responses in monocytic myeloid-derived suppressor cells,which was paired with their pronounced expansion. These results provide novel mechanistic arguments as to why AES may exert context-specific beneficial or adverse effects in patients with critical illness. This study lays out an analytical framework to distill high-dimensional datasets gathered in an interventional clinical trial into a fairly simple model that converges with known biology and provides insight into novel and clinically relevant cellular mechanisms. View Publication