技术资料

Generating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However,the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study,we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly,the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore,the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo. View Publication

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE,which encodes neutrophil elastase (NE). However,a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end,we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs),and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest,and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly,high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBP$$-dependent emergency granulopoiesis. In contrast,sivelestat,an NE-specific small-molecule inhibitor,corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA,but not CEBPB; and promoting promyelocyte survival and differentiation. Together,these data suggest that SCN disease pathogenesis includes NE mislocalization,which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization. View Publication

Many acute and chronic anaemias,including haemolysis,sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia,are not treatable with erythropoietin (Epo),because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently,we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor,burst-forming unit erythroid (BFU-E),and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors,with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis,PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally,both in control and corticosteroid-treated BFU-E cells,PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists,additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPAR-α agonists we used may improve the efficacy of corticosteroids in treating Epo-resistant anaemias. View Publication

Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo,yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5,an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore,Ncx1 heartbeat mutants,as well as static cultures of AGM,exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures,transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. View Publication

BACKGROUND Anagrelide is a cytoreductive agent used to lower platelet counts in essential thrombocythemia. Although the drug has been known to selectively inhibit megakaryopoiesis for many years,the molecular mechanism accounting for this activity is still unclear. OBJECTIVES AND METHODS To address this issue we have compared the global gene expression profiles of human hematopoietic cells treated ex-vivo with and without anagrelide while growing under megakaryocyte differentiation conditions,using high-density oligonucleotide microarrays. Gene expression data were validated by the quantitative polymerase chain reaction and mined to identify functional subsets and regulatory pathways. RESULTS We identified 328 annotated genes differentially regulated by anagrelide,including many genes associated with platelet functions and with the control of gene transcription. Prominent among the latter was TRIB3,whose expression increased in the presence of anagrelide. Pathway analysis revealed that anagrelide up-regulated genes that are under the control of the transcription factor ATF4,a known TRIB3 inducer. Notably,immunoblot analysis demonstrated that anagrelide induced the phosphorylation of eIF2α,which is an upstream regulator of ATF4,and increased ATF4 protein levels. Furthermore,salubrinal,an inhibitor of eIF2α dephosphorylation,increased the expression of ATF4-regulated genes and blocked megakaryocyte growth. CONCLUSIONS These findings link signaling through eIF2α/ATF4 to the anti-megakaryopoietic activity of anagrelide and identify new potential modulators of megakaryopoiesis. 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

The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However,the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here,we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC),an extracellular matrix protein associated with HSC niches,strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders,including the formation of VE-Cadherin(+)CD73(-)CD235a/CD43(-) HE and hematopoietic progenitors with myeloid and T lymphoid potential. View Publication

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with $$-thalassemia ($$-thal) with the aim to generate trangene-free $$-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4,Klf4,Sox2,cMyc,and Lin28 resulted in formation of five iPSC colonies,from which three were picked up and expanded in $$-thal-iPSC lines. After 10 serial passages in vitro,$$-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs,whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%,but with a decreased hematopoietic colony-forming capability. In conclusion,we report herein the generation of transgene-free $$-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover,it was demonstrated that the mRNA-based reprogramming method,used mainly in fibroblasts,is also suitable for reprogramming of human BM-MSCs. View Publication

Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovascular,hematological,and neurological complications. A robust patient-derived cellular model is necessary to investigate the pathophysiology of the syndrome because current animal models are limited and access to tissues from affected individuals is ethically challenging. We aimed to derive induced pluripotent stem cells (iPSCs) from trisomy 21 human mid-trimester amniotic fluid stem cells (AFSCs) and describe their hematopoietic and neurological characteristics. Human AFSCs collected from women undergoing prenatal diagnosis were selected for c-KIT(+) and transduced with a Cre-lox-inducible polycistronic lentiviral vector encoding SOX2,OCT4,KLF-4,and c-MYC (50,000 cells at a multiplicity of infection (MOI) 1-5 for 72 h). The embryonic stem cell (ESC)-like properties of the AFSC-derived iPSCs were established in vitro by embryoid body formation and in vivo by teratoma formation in RAG2(-/-),$\$-chain(-/-),C2(-/-) immunodeficient mice. Reprogrammed cells retained their cytogenetic signatures and differentiated into specialized hematopoietic and neural precursors detected by morphological assessment,immunostaining,and RT-PCR. Additionally,the iPSCs expressed all pluripotency markers upon multiple rounds of freeze-thawing. These findings are important in establishing a patient-specific cellular platform of trisomy 21 to study the pathophysiology of the aneuploidy and for future drug discovery. View Publication

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