技术资料

Gfi-1B is a transcriptional repressor that is crucial for erythroid differentiation: inactivation of the GFI1B gene in mice leads to embryonic death due to failure to produce differentiated red cells. Accordingly,GFI1B expression is tightly regulated during erythropoiesis,but the mechanisms involved in such regulation remain partially understood. We here identify HMGB2,a high-mobility group HMG protein,as a key regulator of GFI1B transcription. HMGB2 binds to the GFI1B promoter in vivo and up-regulates its trans-activation most likely by enhancing the binding of Oct-1 and,to a lesser extent,of GATA-1 and NF-Y to the GFI1B promoter. HMGB2 expression increases during erythroid differentiation concomitantly to the increase of GfI1B transcription. Importantly,knockdown of HMGB2 in immature hematopoietic progenitor cells leads to decreased Gfi-1B expression and impairs their erythroid differentiation. We propose that HMGB2 potentiates GATA-1-dependent transcription of GFI1B by Oct-1 and thereby controls erythroid differentiation. View Publication

The regulatory pathways necessary for the maintenance of adult hematopoietic stem cells (HSCs) remain poorly defined. By using loss-of-function approaches,we report a selective and cell-autonomous requirement for the p300/CBP-binding transcriptional coactivator Cited2 in adult HSC maintenance. Conditional deletion of Cited2 in the adult mouse results in loss of HSCs causing multilineage bone marrow failure and increased lethality. In contrast,conditional ablation of Cited2 after lineage specification in lymphoid and myeloid lineages has no impact on the maintenance of these lineages. Additional deletion of Ink4a/Arf (encoding p16(Ink4a) and p19(Arf)) or Trp53 (encoding p53,a downstream target of p19(Arf)) in a Cited2-deficient background restores HSC functionality and rescues mice from bone marrow failure. Furthermore,we show that the critical role of Cited2 in primitive hematopoietic cells is conserved in humans. Taken together,our studies provide genetic evidence that Cited2 selectively maintains adult HSC functions,at least in part,via Ink4a/Arf and Trp53. View Publication

Since mouse embryonic stem (ES) cells was first derived in 1981,the ability of this unprecedented cell type to self-renew and differentiate without limit has revolutionized the discovery tools that are used to study gene functions and development. Furthermore,they have inspired others to hunt for similar cells from other species. The derivation of human ES cells in 1998 has accelerated these discoveries and has also widely provoked public interest,due to both the scientific significance of these cells for human tissue regeneration and the ethical disputes over the use of donated early human embryos. However,this is no longer a barrier,with the recent discovery of methods that can convert differentiated somatic cells into ES-like cells or induced pluripotent stem (iPS) cells,by using defined reprogramming factors. This review attempts to summarize the progresses in the derivation of ES cells (as well as other embryo-derived pluripotent cells) and iPS cells from various species. We will focus on the molecular and biological features of the cells,as well as the different determinants identified thus far to sustain their pluripotency. View Publication

Autologous feeder cells have been developed by various methods to minimize the presence of xenogenic entities in human embryonic stem cell (hESC) cultures. However,there was no systematic comparison of supportive effects of the feeder cells on hESC growth,nor comparison to the supportive effects of various feeder-free culture systems and standard mouse feeder cells. In this study,we aimed to compare the supportive abilities of autologous feeders derived either directly from H9 hESCs (H9 dF) or from outgrowth of embryoid body predifferentiated in suspension from H9 hESCs (H9 ebF). Mouse feeder system and matrigel-mTeSR1 feeder-free system were used as controls. H9 ebF was found to secrete more basic fibroblast growth factor in the conditioned medium than H9 dF did. The undifferentiated state of H9 hESCs was sustained more stably on H9 ebF than on H9 dF,and the differentiation potential of H9 hESCs on H9 ebF was higher than on H9 dF. We concluded that H9 ebF was an optimal autologous feeder to maintain the long-term undifferentiated state of hESCs in our current culture system. This study helps to standardize the autologous culture of hESCs. It also suggests a more definite direction for future development of xeno-free culture system for hESCs. View Publication

Phenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture,better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs),we demonstrated that the rhodamine-low phenotype was lost,whereas AC133 expression was retained throughout culture. Furthermore,the AC133(+)CD38(-) subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number,and limiting dilution analysis in NOD/SCID mice,showed a 43-fold expansion of the AC133(+)CD38(-) subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation,respectively. Thus,AC133(+)CD38(-) is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures. 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

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

RATIONALE: Studies have demonstrated that bone marrow-derived cells can be recruited to injured lungs through an unknown mechanism. We hypothesize that marrow progenitors are mobilized into the circulation of patients with cardiac and/or respiratory failure,and may then traffic to and incorporate into the sites of tissue injury. OBJECTIVES: To determine whether progenitor populations are increased in the blood of patients with severe acute cardiorespiratory failure placed on extracorporeal membrane oxygenation (ECMO). METHODS: Mononuclear cells from ECMO,umbilical cord,and control blood samples were evaluated in colony-forming assays for hematopoietic,mesenchymal,and epithelial cells. Progenitors were identified by proliferative and differentiative capacities,and confirmed by the expression of lineage-specific markers. MEASUREMENTS AND MAIN RESULTS: Significantly higher levels of hematopoietic progenitors were observed in ECMO (n = 41) samples than neonatal intensive care unit (n = 16) or pediatric intensive care unit controls (n = 14). Hematopoietic progenitor mobilization increased with time on ECMO support. Mesenchymal progenitors (MSC) were recovered from 18/58 ECMO samples with rapid sample processing (textless 4 h) critical to their recovery. MSC were not recovered from normal controls. ECMO-derived MSC had osteogenic,chondrogenic,and adipogenic differentiation potential. The recovery of MSC did not influence survival outcome (61%). Epithelial progenitors were observed in eight ECMO samples but not in control samples. Their presence was associated with a lower survival trend (38%). CONCLUSIONS: Hematopoietic,mesenchymal,and epithelial progenitors were mobilized into the circulation of patients on ECMO. This may reflect a response to severe cardiopulmonary injury,blood-foreign surface interactions with the ECMO circuit,and/or hemodilution. View Publication

Hairy enhancer of split 1 (Hes1) is a basic helix-loop-helix transcriptional repressor that affects differentiation and often helps maintain cells in an immature state in various tissues. Here we show that retroviral expression of Hes1 immortalizes common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in the presence of interleukin-3,conferring permanent replating capability on these cells. Whereas these cells did not develop myeloproliferative neoplasms when intravenously administered to irradiated mice,the combination of Hes1 and BCR-ABL in CMPs and GMPs caused acute leukemia resembling blast crisis of chronic myelogenous leukemia (CML),resulting in rapid death of the recipient mice. On the other hand,BCR-ABL alone caused CML-like disease when expressed in c-Kit-positive,Sca-1-positive,and lineage-negative hematopoietic stem cells (KSLs),but not committed progenitors CMPs or GMPs,as previously reported. Leukemic cells derived from Hes1 and BCR-ABL-expressing CMPs and GMPs were more immature than those derived from BCR-ABL-expressing KSLs. Intriguingly,Hes1 was highly expressed in 8 of 20 patients with CML in blast crisis,but not in the chronic phase,and dominant negative Hes1 retarded the growth of some CML cell lines expressing Hes1. These results suggest that Hes1 is a key molecule in blast crisis transition in CML. View Publication

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases,neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore,midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally,a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression,or by BH3-mimetics such as obatoclax,may be an attractive therapy concept in SM. View Publication

We observed that BMSCs (bone marrow stromal cells) from myeloma patients (myeloma BMSCs) were significantly stiffer than control BMSCs using a cytocompression device. The stiffness of myeloma BMSCs and control BMSCs was further increased upon priming by myeloma cells. Additionally,myeloma cells became stiffer when primed by myeloma BMSCs. The focal adhesion kinase activity of myeloma cells was increased when cells were on stiffer collagen gels and on myeloma BMSCs. This change in myeloma stiffness is associated with increased colony formation of myeloma cells and FAK activation when co-cultured with stiffer myeloma BMSCs or stiffer collagen. Additionally,stem cells of RPMI8226 cells became stiffer after priming by myeloma BMSCs,with concomitant increases of stem cell colony formation. These results suggest the presence of a mechanotransduction loop between myeloma cells and myeloma BMSCs to increase the stiffness of both types of cells via FAK activation. The increase of stiffness may in turn support the growth of myeloma cells and myeloma stem cells. View Publication

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties,we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR,a fluorescent substrate for ALDH,and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56(+) fraction in those cells,but,we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly,ALDH activity and Aldh1a1 expression levels were very low in mouse,rat,rabbit and non-human primate myoblasts. Using different approaches,from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde,an inhibitor of class I ALDH,to cell fractionation by flow cytometry using the ALDEFLUOR assay,we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H(2) O(2) )-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity,as a purification strategy,could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. View Publication