技术资料

The use of human pluripotent stem cells,including embryonic and induced pluripotent stem cells,in therapeutic applications will require the development of robust,scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs),but challenges specific to hESCs will also have to be addressed,including development of defined,humanized culture media and substrates,monitoring spontaneous differentiation and heterogeneity in the cultures,and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. View Publication

Autophagy that is induced by starvation or cellular stress can enable cancer cell survival by sustaining energy homeostasis and eliminating damaged organelles and proteins. In response to stress,cancer cells have been reported to accumulate the protein p62/SQSTM1 (p62),but its role in the regulation of autophagy is controversial. Here,we report that the plant phytoalexin resveratrol (RSV) triggers autophagy in imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia (CML) cells via JNK-dependent accumulation of p62. JNK inhibition or p62 knockdown prevented RSV-mediated autophagy and antileukemic effects. RSV also stimulated AMPK,thereby inhibiting the mTOR pathway. AMPK knockdown or mTOR overexpression impaired RSV-induced autophagy but not JNK activation. Lastly,p62 expression and autophagy in CD34+ progenitors from patients with CML was induced by RSV,and disrupting autophagy protected CD34+ CML cells from RSV-mediated cell death. We concluded that RSV triggered autophagic cell death in CML cells via both JNK-mediated p62 overexpression and AMPK activation. Our findings show that the JNK and AMPK pathways can cooperate to eliminate CML cells via autophagy. View Publication

The immunodeficiency disorder,X-linked agammaglobulinemia (XLA),results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA,we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice,a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells,treated mice showed significant,albeit incomplete,rescue of mature B cells in the bone marrow,peripheral blood,spleen,and peritoneal cavity,and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression,viral integration,and partial functional responses,consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA. View Publication

Clonal analysis is important for many areas of hematopoietic stem cell research,including in vitro cell expansion,gene therapy,and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle,they generally provide a low-resolution,biased,and incomplete assessment of clonality. To overcome those limitations,we labeled retroviral vectors with random sequence tags or barcodes." On integration� View Publication

Human embryonic stem (hES) cells have enormous potential for clinical applications. However,one major challenge is to achieve high cell recovery rate after cryopreservation. Understanding how the conventional cryopreservation protocol fails to protect the cells is a prerequisite for developing efficient and successful cryopreservation methods for hES cell lines and banks. We investigated how the stimuli from cryopreservation result in apoptosis,which causes the low cell recovery rate after cryopreservation. The level of reactive oxygen species (ROS) is significantly increased,F-actin content and distribution is altered,and caspase-8 and caspase-9 are activated after cryopreservation. p53 is also activated and translocated into nucleus. During cryopreservation apoptosis is induced by activation of both caspase-8 through the extrinsic pathway and caspase-9 through the intrinsic pathway. However,exactly how the extrinsic pathway is activated is still unclear and deserves further investigation. View Publication

Meticulous characterization of human embryonic stem cells (hESC) is critical to their eventual use in cell-based therapies,particularly in view of the diverse methods for derivation and maintenance of these cell lines. However,characterization methods are generally not standardized and many currently used assays are subjective,making dependable and direct comparison of cell lines difficult. In order to address this problem,we selected 10 molecular-based high-resolution assays as components of a panel for characterization of hESC. The selection of the assays was primarily based on their quantitative or objective (rather than subjective) nature. We demonstrate the efficacy of this panel by characterizing 4 hESC lines,derived in two different laboratories using different derivation techniques,as pathogen free,genetically stable,and able to differentiate into derivatives of all three germ layers. Our panel expands and refines a characterization panel previously proposed by the International Stem Cell Initiative and is another step toward standardized hESC characterization and quality control,a crucial element of successful hESC research and clinical translation. View Publication

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can be induced to differentiate into myogenic cells. Despite their potential,previous studies have not been successful in producing a high percentage of cardiac-like cells with a muscle phenotype. We hypothesized that cardiac lineage development in BM-MSC is related to cell passage,culture milieu,and enrichment for specific cell subtypes before and during differentiation. Our study demonstrated that Lin(-) BM-MSC at an intermediate passage (IP; P8-P12) expressed cardiac troponin T (cTnT) after 21 days in culture. Cardiac TnT expression was similar whether IP cells were differentiated in media containing 5-azacytidine+2% FBS (AZA; 14%) or 2% FBS alone (LS; 12%) and both were significantly higher than AZA+5% FBS. This expression was potentiated by first enriching for CD117/Sca-1 cells followed by differentiation (AZA,39% and LS,28%). A second sequential enrichment for the dihydropyridine receptor subunit alpha2delta1 (DHPR-alpha2) resulted in cardiac TnT expressed in 54% of cultured cells compared to 28% of cells after CD117/Sca-1(+) enrichment. Cells enriched for CD117/Sca-1 and subjected to differentiation displayed spontaneous intracellular Ca(2+) transients with an increase in transient frequency and a 60% decrease in the transient duration amplitude between days 14 and 29. In conclusion,IP CD117/Sca-1(+) murine BM-MSCs display robust cardiac muscle lineage development that can be induced independent of AZA but is diminished under higher serum concentrations. Furthermore,temporal changes in calcium kinetics commensurate with increased cTnT expression suggest progressive maturation of a cardiac muscle lineage. Enrichment with CD117/Sca-1 to establish lineage commitment followed by DHPR-alpha2 in lineage developing cells may enhance the therapeutic potential of these cells for transplantation. View Publication

BACKGROUND: Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However,the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product,both of which can limit the future clinical application of hESC-ECs. Moreover,to fully understand the beneficial effects of stem cell therapy,investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time. METHODOLOGY: In this study,we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray,and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover,our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods. CONCLUSION: Taken together,we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes,form functional vessels in vivo,and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future. View Publication

The Tal1 transcription factor is essential for the development of the hematopoietic system and plays a role during definitive erythropoiesis in the adult. Despite the importance of Tal1 in erythropoiesis,only a small number of erythroid differentiation target genes are known. A chromatin precipitation and cloning approach was established to uncover novel Tal1 target genes in erythropoiesis. The BirA tag/BirA ligase biotinylation system in combination with streptavidin chromatin precipitation (Strep-CP) was used to co-precipitate genomic DNA bound to Tal1. Tal1 was found to bind in the vicinity of 31 genes including the E2-ubiquitin conjugase UBE2H gene. Binding of Tal1 to UBE2H was confirmed by chromatin immunoprecipitation. UBE2H expression is increased during erythroid differentiation of hCD34(+) cells. Tal1 expression activated UBE2H expression,whereas Tal1 knock-down reduced UBE2H expression and ubiquitin transfer activity. This study identifies parts of the ubiquitinylation machinery as a cellular target downstream of the transcription factor Tal1 and provides novel insights into Tal1-regulated erythropoiesis. View Publication

Due to widespread applications of human embryonic stem (hES) cells,it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation,and further investigated the role of the combination of Rho-associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow-freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture,we found out that hES cell recovery was significantly enhanced by around 30 % (P textless 0.05) by the new freezing solution. Moreover,at the first day of post-thaw culture,the presence of 10 microM ROCK inhibitor (Y-27632) and 1 microM pifithrin-mu together further significantly improved cell recovery by around 20% (P textless 0.05) either for feeder-dependent or feeder-independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore,this protocol is a scalable cryopreservation method for handling large quantities of hES cells. View Publication

Neonatal hyperoxia impairs vascular and alveolar growth in mice and decreases endothelial progenitor cells. To determine the role of bone marrow-derived cells in restoration of neonatal lung structure after injury,we studied a novel bone marrow myeloid progenitor cell population from Tie2-green fluorescent protein (GFP) transgenic mice (bone marrow-derived angiogenic cells; BMDAC). We hypothesized that treatment with BMDAC would restore normal lung structure in infant mice during recovery from neonatal hyperoxia. Neonatal mice (1-day-old) were exposed to 80% oxygen for 10 days. BMDACs (1 x 10(5)),embryonic endothelial progenitor cells,mouse embryonic fibroblasts (control),or saline were then injected into the pulmonary circulation. At 21 days of age,saline-treated mice had enlarged alveoli,reduced septation,and a reduction in vascular density. In contrast,mice treated with BMDAC had complete restoration of lung structure that was indistinguishable from room air controls. BMDAC comprised 12% of distal lung cells localized to pulmonary vessels or alveolar type II (AT2) cells and persist (8.8%) for 8 wk postinjection. Coculture of AT2 cells or lung endothelial cells (luEC) with BMDAC augmented AT2 and luEC cell growth in vitro. We conclude that treatment with BMDAC after neonatal hyperoxia restores lung structure in this model of bronchopulmonary dysplasia. View Publication

We report an injectable hydrogel scaffold system with tunable stiffness for controlling the proliferation rate and differentiation of human mesenchymal stem cells (hMSCs) in a three-dimensional (3D) context in normal growth media. The hydrogels composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugate were formed using the oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H(2)O(2)) and horseradish peroxidase (HRP). The stiffness of the hydrogels was readily tuned by varying the H(2)O(2) concentration without changing the concentration of polymer precursor. We found that the hydrogel stiffness strongly affected the cell proliferation rates. The rate of hMSC proliferation increased with the decrease in the stiffness of the hydrogel. Also,the neurogenesis of hMSCs was controlled by the hydrogel stiffness in a 3D context without the use of any additional biochemical signal. These cells which were cultured in hydrogels with lower stiffness for 3 weeks expressed much more neuronal protein markers compared to those cultured within stiffer hydrogels for the same period of time. View Publication