技术资料

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However,attempts to isolate MSCs from umbilical cord blood (UCB) of full-term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria. Using optimized isolation and culture conditions,in up to 63% of 59 low-volume UCB units,cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) were isolated. These were similar to control MSCs from adult bone marrow (BM). The frequency of MSC-like cells ranged from 0 to 2.3 clones per 1 x 10(8) mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition,osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However,in contrast to MSCs,MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation. Crucial points to isolate MSC-like cells from UCB were a time from collection to isolation of less than 15 hours,a net volume of more than 33 ml,and an MNC count of more than 1 x 10(8) MNCs. Because MSC-like cells can be isolated at high efficacy from full-term UCB donations,we regard UCB as an additional stem cell source for experimental and potentially clinical purposes. View Publication

The Kit receptor functions in hematopoiesis,lymphocyte development,gastrointestinal tract motility,melanogenesis,and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo,we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice,lymphopoiesis is affected differentially. The KitY567F mutation,but not the KitY719F mutation,blocks pro T cell and pro B cell development in an age-dependent manner. Thus,the Src family kinase,but not the PI 3-kinase docking site in Kit,mediates a critical signal for lymphocyte development. In agreement with these results,treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development,similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore,Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo. View Publication

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors,namely insulin receptor substrate-2 (IRS2),Src homology 2 domain-containing inositol 5'-phosphatase (SHIP),Grb2-associated binder-1 (Gab1),and the Epo receptor (EpoR). Using different in vitro systems,we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors,Akt,FKHRL1,and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR),through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR,or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors,but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors,the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally,our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2),which,in turn,down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication

The chicken anemia virus-derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However,the mechanisms through which apoptin achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein-apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S,and the leukemia cell lines K562,HL60,U937,KG1,and NB4. In contrast,normal CD34(+) cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition,dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases,protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed,shRNA knockdown or drug-mediated inhibition of PKCβ significantly reduced apoptin phosphorylation. Furthermore,apoptin-mediated cell death proceeded through the upregulation of PKCβ,activation of caspase-9/3,cleavage of the PKCδ catalytic domain,and downregulation of the MERTK and AKT kinases. Collectively,these results elucidate a novel pathway for apoptin activation involving PKCβ and PKCδ. Further,they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma. View Publication

The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently,human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However,the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages,including enabling production of larger scale lots,decreasing lead time to generate purified iPSC-BMEC cultures,and facilitating use of iPSC-BMECs in large-scale screening. In this study,we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability,express standard endothelial and BBB markers,and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ωtextperiodcenteredcm(2),equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs,as well as stabilized TEER above 800 Ωtextperiodcenteredcm(2) out to 7 days post-thaw. Overall,cryopreservation will ease handling and storage of high-quality iPSC-BMECs,reducing a key barrier to greater implementation of these cells in modeling the human BBB. View Publication

Cryopreservation is an essential technique to preserve stem cells,semipermanently sustaining their potentials. There are two main approaches of cryopreservation for human pluripotent stem cells (hPSCs). The first is the vitrification,which involves instantaneous freeze and thaw of hPSCs. The second is the conventional slow-cooling method and a rapid thaw. Both cryopreservation protocols have been standardized and optimized to yield high survivability of hPSCs. View Publication

BACKGROUND Cryopreservation protocols have remained relatively unchanged since the first umbilical cord blood banking program was established. This study evaluated the preservation efficacy of a novel intracellular-like cryopreservation solution (CryoStor,BioLife Solutions,Inc.),the rate of addition of two cryopreservation solutions to cord blood units (CBUs),and reduced final dimethyl sulfoxide (DMSO) concentration of 5%. STUDY DESIGN AND METHODS Split-sample CBUs were cryopreserved with either an in-house 20% DMSO-based cryopreservation solution or CryoStor CS10 at a rate of 1 mL/min (n = 10; i.e.,slow addition) or as a bolus injection (n = 6; i.e.,fast addition). Infrared images of exothermic effects of the cryopreservation solutions were monitored relative to the rate of addition. Prefreeze and postthaw colony-forming unit assays,total nucleated cells,and CD34+ cell counts were compared. RESULTS Maximum temperature excursions observed were less than 6°C,regardless of the rate of solution addition. Fast addition resulted in peak excursions approximately twice that of slow addition but the magnitude and duration were minimal and transient. Slow addition of CryoStor CS10 (i.e.,final concentration % 5% DMSO) resulted in significantly better postthaw CD34+ cell recoveries; no other metrics were significantly different. Fast addition of CryoStor resulted in similar postthaw metrics compared to slow addition of the in-house solution. CONCLUSION Slow and fast addition of cryopreservation solutions result in mean temperature changes of approximately 3.3 to 4.45°C. Postthaw recoveries with CryoStor were equivalent to or slightly better than with the in-house cryopreservation solution. CryoStor also provides several advantages including reduced processing time,formulation consistency,and reduced DMSO in the frozen product (% 5%). View Publication

Clinical trials are currently used to test therapeutic efficacies for lung cancer,infections and diseases. Animal models are also used as surrogates for human disease. Both approaches are expensive and time-consuming. The utility of human biospecimens as models is limited by specialized tissue processing methods that preserve subclasses of analytes (e.g. RNA,protein,morphology) at the expense of others. We present a rapid and reproducible method for the cryopreservation of viable lung tissue from patients undergoing lobectomy or transplant. This method involves the pseudo-diaphragmatic expansion of pieces of fresh lung tissue with cryoprotectant formulation (pseudo-diaphragmatic expansion-cryoprotectant perfusion or PDX-CP) followed by controlled-rate freezing in cryovials. Expansion-perfusion rates,volumes and cryoprotectant formulation were optimized to maintain tissue architecture,decrease crystal formation and increase long-term cell viability. Rates of expansion of 4 cc/min or less and volumes ranging from 0.8-1.2 × tissue volume were well-tolerated by lung tissue obtained from patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis,showing minimal differences compared to standard histopathology. Morphology was greatly improved by the PDX-CP procedure compared to simple fixation. Fresh versus post-thawed lung tissue showed minimal differences in histology,RNA integrity numbers and post-translational modified protein integrity (2-dimensional differential gel electrophoresis). It was possible to derive numerous cell types,including alveolar epithelial cells,fibroblasts and stem cells,from the tissue for at least three months after cryopreservation. This new method should provide a uniform,cost-effective approach to the banking of biospecimens,with versatility to be amenable to any post-acquisition process applicable to fresh tissue samples. View Publication