技术资料

Granulocyte colony-stimulating factor (G-CSF),the prototypical mobilizing cytokine,induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated,in part,through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization,osteoblast suppression,and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact,demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover,G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally,we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together,these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance,ultimately leading to HSPC mobilization. View Publication

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects,C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover,in K562 cells,RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1,but not of a transcriptional repressor mutant (Gfi-1P2A),inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast,Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together,these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. View Publication

BACKGROUND: Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood,as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow,detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al,2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens,with particular attention to the expression of ALDH on erythroid precursors. To this aim,we used three kinds of approach: i) multidimensional analytical flow cytometry,detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells,followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. RESULTS: In normal bone marrow,we identified three populations of cells,namely ALDH+CD34+,ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52,0.53 and 0.57,respectively). As compared to ALDH-CD34+ cells,ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells,with brighter expression of CD117 and CD133,accompanied by lower display of CD38 and CD45RA. Of interest,ALDH+CD34- population disclosed a straightforward erythroid commitment,on the basis of three orders of evidences. First of all,ALDH+CD34- cells showed a CD71bright,CD105+,CD45- phenotype. Secondly,induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally,ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). CONCLUSION: Our study,comparing surface antigen expression of ALDH+/CD34+,ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow,clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia. View Publication

Human embryonic stem (hES) cells hold great promise for application of human cell and tissue replacement therapy. However,the overwhelming majority of currently available hES cell lines have been directly or indirectly exposed to materials containing animal-derived components during their derivation,propagation,and cryopreservation. Unlike feeder based cultures,which require the simultaneous growth of feeder and stem cells,resulting in mixed cell populations,stem cells grown on feeder-free systems are easily separated from the surface,presenting a pure population of cells for downstream applications. In this study we have developed a novel method to expand hES cells in xeno-free,feeder-free conditions using two different matrices derived from xeno-free human foreskin fibroblasts (XF-HFFs). Using XF-HFF-derived extracellular matrix,together with 100ng/ml recombinant bFGF supplemented HEScGRO Basal Medium,long term xeno-free expansion of hES cells is possible. Resulting hES cells were subjected to stringent tests and were found to maintain ES cell features,including morphology,pluripotency,stable karyotype,and expression of cell surface markers,for at least 20 passages. Xeno-free culturing practices are essential for the translation of basic hES cell research into the clinic. Therefore,the method presented in this study demonstrates that hES cells can be cultured in complete xeno-free conditions without the loss of pluripotency and furthermore,without the possibility of contamination from exogenous sources. View Publication

Although extracellular nucleotides support a wide range of biologic responses of mature blood cells,little is known about their effect on blood cell progenitor cells. In this study,we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs),and whether activation by their natural ligands,adenosine triphosphate (ATP) and uridine triphosphate (UTP),induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34(+) HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore,stimulation of CD34(+) cells with extracellular nucleotides caused a fast release of Ca(2+) from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally,ATP and,to a higher extent,UTP acted as potent early acting growth factors for HSCs,in vitro,because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34(+) and lineage-negative CD34(-) progenitors and expanded more primitive CD34(+)-derived long-term culture-initiating cells. Furthermore,xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions. View Publication

Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level,it results in drastic chromatin changes to erase the existing somatic memory" and to establish the pluripotent state. Accordingly� View Publication

The incidence of cardiovascular disease represents a significant and growing health-care challenge to the developed and developing world. The ability of native heart muscle to regenerate in response to myocardial infarct is minimal. Tissue engineering and regenerative medicine approaches represent one promising response to this difficulty. Here,we present methods for the construction of a cell-seeded cardiac patch with the potential to promote regenerative outcomes in heart muscle with damage secondary to myocardial infarct. This method leverages iPS cells and a fibrin-based scaffold to create a simple and commercially viable tissue-engineered cardiac patch. Human-induced pluripotent stem cells (hiPSCs) can,in principle,be differentiated into cells of any lineage. However,most of the protocols used to generate hiPSC-derived endothelial cells (ECs) and cardiomyocytes (CMs) are unsatisfactory because the yield and phenotypic stability of the hiPSC-ECs are low,and the hiPSC-CMs are often purified via selection for expression of a promoter-reporter construct. In this chapter,we describe an hiPSC-EC differentiation protocol that generates large numbers of stable ECs and an hiPSC-CM differentiation protocol that does not require genetic manipulation,single-cell selection,or sorting with fluorescent dyes or other reagents. We also provide a simple but effective method that can be used to combine hiPSC-ECs and hiPSC-CMs with hiPSC-derived smooth muscle cells to engineer a contracting patch of cardiac cells. View Publication

The generation of liquefied poly-ɛ-caprolactone (PCL) droplets by means of a microfluidic device results in uniform-sized microspheres,which are validated as microcarriers for human embryonic stem cell culture. Formed droplet size and size distribution,as well as the resulting PCL microsphere size,are correlated with the viscosity and flow rate ratio of the dispersed (Q d) and continuous (Q c) phases. PCL in dichloromethane increases its viscosity with concentration and molecular weight. Higher viscosity and Q d/Q c lead to the formation of larger droplets,within two observed formation modes: dripping and jetting. At low viscosity of dispersed phase and Q d/Q c,the microfluidic device is operated in dripping mode,which generates droplets and microspheres with greater size uniformity. Solutions with lower molecular weight PCL have lower viscosity,resulting in a wider concentration range for the dripping mode. When coated with extracellular matrix (ECM) proteins,the fabricated PCL microspheres are demonstrated capable of supporting the expansion of human embryonic stem cells. View Publication

A prerequisite for the realization of human pluripotent stem cell (hPSC) therapies is the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable expansion and directed differentiation of hPSCs. Comparative analyses of commercially available microcarriers clearly show the need for developing synthetic substrates supporting the adhesion and growth of hPSCs in three-dimensional cultures under agitation-induced shear. Moreover,the low seeding efficiencies during microcarrier loading with hPSC clusters poses a significant process bottleneck. To that end,a novel protocol was developed increasing hPSC seeding efficiency from 30% to over 80% and substantially shortening the duration of microcarrier loading. Importantly,this method was combined with the engineering of polystyrene microcarriers by surface conjugation of a vitronectin-derived peptide,which was previously shown to support the growth of human embryonic stem cells. Cells proliferated on peptide-conjugated beads in static culture but widespread detachment was observed after exposure to stirring. This prompted additional treatment of the microcarriers with a synthetic polymer commonly used to enhance cell adhesion. hPSCs were successfully cultivated on these microcarriers in stirred suspension vessels for multiple consecutive passages with attachment efficiencies close to 40%. Cultured cells exhibited on average a 24-fold increase in concentration per 6-day passage,over 85% viability,and maintained a normal karyotype and the expression of pluripotency markers such as Nanog,Oct4,and SSEA4. When subjected to spontaneous differentiation in embryoid body cultures or directed differentiation to the three embryonic germ layers,the cells adopted respective fates displaying relevant markers. Lastly,engineered microcarriers were successfully utilized for the expansion and differentiation of hPSCs to mesoderm progeny in stirred suspension vessels. Hence,we demonstrate a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable,xeno-free propagation and differentiation of human stem cells intended for therapies. 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

Signal transduction mediated by Fas-associated death domain protein (FADD) represents a paradigm of coregulation of apoptosis and cellular proliferation. During apoptotic signaling induced by death receptors including Fas,FADD is required for the recruitment and activation of caspase 8. In addition,a death receptor-independent function of FADD is essential for embryogenesis. In previous studies,FADD deficiency in embryonic stem cells resulted in a complete lack of B cells and dramatically reduced T cell numbers,as shown by Rag1(-/-) blastocyst complementation assays. However,T-specific FADD-deficient mice contained normal numbers of thymocytes and slightly reduced peripheral T cell numbers,whereas B cell-specific deletion of FADD led to increased peripheral B cell numbers. It remains undetermined what impact an FADD deficiency has on hematopoietic stem cells and progenitors. The current study analyzed the effect of simultaneous deletion of FADD in multiple cell types,including bone marrow cells,by using the IFN-inducible Mx1-cre transgene. The resulting FADD mutant mice did not develop lymphoproliferation diseases,unlike Fas-deficient mice. Instead,a time-dependent depletion of peripheral FADD-deficient lymphocytes was observed. In the bone marrow,a lack of FADD led to a dramatic decrease in the hematopoietic stem cells and progenitor-enriched population. Furthermore,FADD-deficient bone marrow cells were defective in their ability to generate lymphoid,myeloid,and erythroid cells. Thus,the results revealed a temporal requirement for FADD. Although dispensable during lymphopoiesis post lineage commitment,FADD plays a critical role in early hematopoietic stages in the bone marrow. View Publication