技术资料

Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood,aside from numerous congenital abnormalities. FA mouse models have been generated; however,they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero,a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology,we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential,disruption of the second allele causes a severe growth disadvantage. As a result,heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele,quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning,this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage. ?? 2014. View Publication

The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However,it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps,we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in beta-hemoglobin gene (HBB) that cause severe beta-thalassemia (beta-Thal),corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting,and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing,we uncovered seven copy number variations,five small insertions/deletions,and 64 single nucleotide variations (SNVs) in beta-Thal iPSCs before the gene targeting step and found a single small copy number variation,19 insertions/deletions,and 340 single nucleotide variations in the final gene-corrected beta-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps,suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting. View Publication

The clinical use of human embryonic stem cells (hESCs) requires efficient cellular expansion that must be paired with an ability to generate specialized progeny through differentiation. Self-renewal and differentiation are deemed inherent hallmarks of hESCs and a growing body of evidence suggests that initial culture conditions dictate these two aspects of hESC behavior. Here,we reveal that defined culture conditions using commercial mTeSR1 media augment the expansion of hESCs and enhance their capacity for neural differentiation at the expense of hematopoietic lineage competency without affecting pluripotency. This culture-induced modification was shown to be reversible,as culture in mouse embryonic fibroblast-conditioned media (MEF-CM) in subsequent passages allowed mTeSR1-expanded hESCs to re-establish hematopoietic differentiation potential. Optimal yield of hematopoietic cells can be achieved by expansion in mTeSR1 followed by a recovery period in MEF-CM. Furthermore,the lineage propensity to hematopoietic and neural cell types could be predicted via analysis of surrogate markers expressed by hESCs cultured in mTeSR1 versus MEF-CM,thereby circumventing laborious in vitro differentiation assays. Our study reveals that hESCs exist in a range of functional states and balance expansion with differentiation potential,which can be modulated by culture conditions in a predictive and quantitative manner. Stem Cells 2015;33:1142-1152. View Publication

The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs),subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB),and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However,the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study,we used the latest gene-editing tool,clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9),to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then,we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation,gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly,the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system,and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation. View Publication

Without effective therapy,chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized,but biologically poorly characterized,accelerated phase (AP). Here,we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts,which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML,we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML,including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients. View Publication

Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues,including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury,while poor tissue availability of the protein limits its efficacy. Here,we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy)ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine),which selectively activates the tissue-protective EPO receptor,comprising an EPO receptor subunit (EPOR) and the common β-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR,CD131,and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations,STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer,lack of activity in off-target selectivity screening,and lack of functional effects in erythroleukemia cell line TF-1 and CD34(+) progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge,STS-E412 is the first nonpeptidyl,selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted. View Publication

Erythropoietin (Epo) has been used in the treatment of anemia resulting from numerous etiologies,including renal disease and cancer. However,its effects are controversial and the expression pattern of the Epo receptor (Epo-R) is debated. Using in vivo lineage tracing,we document that within the hematopoietic and mesenchymal lineage,expression of Epo-R is essentially restricted to erythroid lineage cells. As expected,adult mice treated with a clinically relevant dose of Epo had expanded erythropoiesis because of amplification of committed erythroid precursors. Surprisingly,we also found that Epo induced a rapid 26% loss of the trabecular bone volume and impaired B-lymphopoiesis within the bone marrow microenvironment. Despite the loss of trabecular bone,hematopoietic stem cell populations were unaffected. Inhibition of the osteoclast activity with bisphosphonate therapy blocked the Epo-induced bone loss. Intriguingly,bisphosphonate treatment also reduced the magnitude of the erythroid response to Epo. These data demonstrate a previously unrecognized in vivo regulatory network coordinating erythropoiesis,B-lymphopoiesis,and skeletal homeostasis. Importantly,these findings may be relevant to the clinical application of Epo. View Publication

HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus,the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here,we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by textgreater10 000-fold for both CD34(+)/CD38(-) and CD34(+)/CD38(+) cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also,we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore,a TAT-SALL4B fusion rapidly expanded CD34(+) cells,and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs. View Publication

The requirement of c-Myb during erythropoiesis spurred an interest in identifying c-Myb target genes that are important for erythroid development. Here,we determined that the neuropeptide neuromedin U (NmU) is a c-Myb target gene. Silencing NmU,c-myb,or NmU's cognate receptor NMUR1 expression in human CD34(+) cells impaired burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) formation compared with control. Exogenous addition of NmU peptide to NmU or c-myb siRNA-treated CD34(+) cells rescued BFU-E and yielded a greater number of CFU-E than observed with control. No rescue of BFU-E and CFU-E growth was observed when NmU peptide was exogenously added to NMUR1 siRNA-treated cells compared with NMUR1 siRNA-treated cells cultured without NmU peptide. In K562 and CD34(+) cells,NmU activated protein kinase C-βII,a factor associated with hematopoietic differentiation-proliferation. CD34(+) cells cultured under erythroid-inducing conditions,with NmU peptide and erythropoietin added at day 6,revealed an increase in endogenous NmU and c-myb gene expression at day 8 and a 16% expansion of early erythroblasts at day 10 compared to cultures without NmU peptide. Combined,these data strongly support that the c-Myb target gene NmU functions as a novel cofactor for erythropoiesis and expands early erythroblasts. View Publication

A defect in cell trafficking and chemotaxis plays an important role in the immune deficiency observed in Wiskott-Aldrich syndrome (WAS). In this report,we show that marrow cells from WAS protein (WASP)-deficient mice also have a defect in chemotaxis. Serial transplantation and competitive reconstitution experiments demonstrated that marrow cells,including hematopoietic progenitors and stem cells (HSCs),have decreased homing capacities that were associated with a defect in adhesion to collagen. During development,HSCs migrate from the liver to the marrow and the spleen,prompting us to ask if a defect in HSC homing during development may explain the skewed X-chromosome inactivation in WAS carriers. Preliminary evidence has shown that,in contrast to marrow progenitor cells,fetal liver progenitor cells from heterozygous females had a random X-chromosome inactivation. When fetal liver cells from WASP-carrier females were injected into irradiated recipients,a nonrandom inactivation of the X-chromosome was found at the level of hematopoietic progenitors and HSCs responsible for the short- and long-term hematopoietic reconstitution. Therefore,the mechanism of the skewed X-chromosomal inactivation observed in WAS carriers may be related to a migration defect of WASP-deficient HSCs. View Publication

BACKGROUND Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential,a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation,the medium for 3D microcarriers was directly switched to EB medium. RESULTS hESCs on 3D microcarriers maintained pluripotency and formed EBs,which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating,EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore,this 3D system can also be adapted to human induced pluripotent stem cells,which generate functional hemangioblasts with high efficiency. CONCLUSION This 3D serum- and stromal-free microcarrier system is important for future clinical applications,with the potential of developing to a GMP-compatible scalable system. View Publication

Human induced pluripotent stem cells (hiPSCs),like embryonic stem cells,are under intense investigation for novel approaches to model disease and for regenerative therapies. Here,we describe the derivation and characterization of hiPSCs from a variety of sources and show that,irrespective of origin or method of reprogramming,hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium,CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage,we were able to demonstrate by single cell analysis (flow cytometry),that hiPSC-derived erythroblasts express alpha globin as previously described,and that a sub-population of these erythroblasts also express haemoglobin F (HbF),indicative of fetal definitive erythropoiesis. More notably however,we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner,but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover,the HbA expressing erythroblast population could be greatly enhanced (44textperiodcentered0 ± 6textperiodcentered04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine. View Publication