技术资料

Cell transplantation has been well explored for cartilage regeneration. We recently showed that the entire articular surface of a synovial joint can regenerate by endogenous cell homing and without cell transplantation. However,the sources of endogenous cells that regenerate articular cartilage remain elusive. Here,we studied whether cytokines not only chemotactically recruit adipose stem cells (ASCs),mesenchymal stem cells (MSCs),and synovium stem cells (SSCs) but also induce chondrogenesis of the recruited cells. Recombinant human transforming growth factor-β3 (TGF-β3; 100 ng) and/or recombinant human stromal derived factor-1β (SDF-1β; 100 ng) was control released into an acellular collagen sponge cube with underlying ASCs,MSCs,or SSCs in monolayer culture. Although all cell types randomly migrated into the acellular collagen sponge cube,TGF-β3 and/or SDF-1β recruited significantly more cells than the cytokine-free control group. In 6 wk,TGF-β3 alone recruited substantial numbers of ASCs (558±65) and MSCs (302±52),whereas codelivery of TGF-β3 and SDF-1β was particularly chemotactic to SSCs (400±120). Proliferation of the recruited cells accounted for some,but far from all,of the observed cellularity. TGF-β3 and SDF-1β codelivery induced significantly higher aggrecan gene expression than the cytokine-free group for ASCs,MSCs,and SSCs. Type II collagen gene expression was also significantly higher for ASCs and SSCs by SDF-1 and TGF-β3 codelivery. Remarkably,the expression of aggrecan and type II collagen was detected among all cell types. Thus,homing of multiple stem/progenitor cell populations may potentially serve as an alternative or adjunctive approach to cell transplantation for cartilage regeneration. View Publication

Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function,but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription,dynamic chromatin architecture,transcription factor binding,and genomic organization in regulation of erythrocyte membrane protein genes,we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly,most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites,located primarily in introns. Cooccupancy with FOG-1,SCL,and MTA-2 was found at all regions of GATA-1 binding,with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4,GATA-1,NF-E2,FOG-1,SCL,and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia. View Publication

Hematopoietic stem cells (HSC) can be harmed by disease,chemotherapy,radiation,and normal aging. We show in this study that damage also occurs in mice repeatedly treated with very low doses of LPS. Overall health of the animals was good,and there were relatively minor changes in marrow hematopoietic progenitors. However,HSC were unable to maintain quiescence,and transplantation revealed them to be myeloid skewed. Moreover,HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor. This phenomenon was previously seen in normal aging. Screening identified mAbs that resolve HSC subsets,and relative proportions of these HSC changed with age and/or chronic LPS treatment. For example,minor CD150(Hi)CD48(-) populations lacking CD86 or CD18 expanded. Simultaneous loss of CD150(Lo/-)CD48(-) HSC and gain of the normally rare subsets,in parallel with diminished transplantation potential,would be consistent with age- or TLR-related injury. In contrast,HSC in old mice differed from those in LPS-treated animals with respect to VCAM-1 or CD41 expression and lacked proliferation abnormalities. HSC can be exposed to endogenous and pathogen-derived TLR ligands during persistent low-grade infections. This stimulation might contribute in part to HSC senescence and ultimately compromise immunity. View Publication

BACKGROUND: Although current evidence suggests that only a minuscule number of osteoblast-lineage cells are present in peripheral blood,we hypothesized that such cells circulate but that their concentration has been vastly underestimated owing to the use of assays that required adherence to plastic. We further reasoned that the concentration of these cells is elevated during times of increased bone formation,such as during pubertal growth. METHODS: We used flow cytometry with antibodies to bone-specific proteins to identify circulating osteoblast-lineage cells in 11 adolescent males and 11 adult males (mean [+/-SD] age,14.5+/-0.7 vs. 37.7+/-7.6 years). Gene expression and in vitro and in vivo bone-forming assays were used to establish the osteoblastic lineage of sorted cells. RESULTS: Cells positive for osteocalcin and cells positive for bone-specific alkaline phosphatase were detected in the peripheral blood of adult subjects (1 to 2 percent of mononuclear cells). There were more than five times as many cells positive for osteocalcin in the circulation of adolescent boys (whose markers of bone formation were clearly increased as a result of pubertal growth) as compared with adult subjects (Ptextless0.001). The percentage of cells positive for osteocalcin correlated with markers of bone formation. Sorted osteocalcin-positive cells expressed osteoblastic genes,formed mineralized nodules in vitro,and formed bone in an in vivo transplantation assay. Increased values were also found in three adults with recent fractures. CONCLUSIONS: Osteoblast-lineage cells circulate in physiologically significant numbers,correlate with markers of bone formation,and are markedly higher during pubertal growth; therefore,they may represent a previously unrecognized circulatory component to the process of bone formation. 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

Cited2 (cAMP-responsive elementbinding protein [CBP]/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]-rich tail 2) is a newly identified transcriptional modulator. Knockout of the Cited2 gene results in embryonic lethality with embryos manifesting heart and neural tube defects. Cited2-/- fetal liver displayed significant reduction in the numbers of Lin(-)c-Kit+Sca-1+ cells,Lin(-)c-Kit+ cells,and progenitor cells of different lineages. Fetal liver cells from Cited2-/- embryos gave rise to markedly reduced number of colonies in the colony-forming unit assay. Primary and secondary transplantation studies showed significantly compromised reconstitution of T-lymphoid,B-lymphoid,and myeloid lineages in mice that received a transplant of Cited2-/- fetal liver cells. Competitive reconstitution experiments further showed that fetal liver hematopoietic stem cell (HSC) function is severely impaired due to Cited2 deficiency. Microarray analysis showed decreased expression of Wnt5a and a panel of myeloid molecular markers such as PRTN3,MPO,Neutrophil elastase,Cathepsin G,and Eosinophil peroxidase in Cited2-/- fetal livers. Decreased expression of Bmi-1,Notch1,LEF-1,Mcl-1,and GATA2 was also observed in Cited2-/- Lin(-)c-Kit+ cells. The present study uncovers for the first time a novel role of Cited2 in the maintenance of hematopoietic homeostasis during embryogenesis and thus provides new insights into the molecular regulation of hematopoietic development. View Publication

There has been a great deal of scientific interest recently generated by the potential therapeutic applications of adult stem cells in human care but there are several challenges regarding quality and safety in clinical applications and a number of these challenges relate to the processing and banking of these cells ex-vivo. As the number of clinical trials and the variety of adult cells used in regenerative therapy increases,safety remains a primary concern. This has inspired many nations to formulate guidelines and standards for the quality of stem cell collection,processing,testing,banking,packaging and distribution. Clinically applicable cryopreservation and banking of adult stem cells offers unique opportunities to advance the potential uses and widespread implementation of these cells in clinical applications. Most current cryopreservation protocols include animal serum proteins and potentially toxic cryoprotectant additives (CPAs) that prevent direct use of these cells in human therapeutic applications. Long term cryopreservation of adult stem cells under good manufacturing conditions using animal product free solutions is critical to the widespread clinical implementation of ex-vivo adult stem cell therapies. Furthermore,to avoid any potential cryoprotectant related complications,reduced CPA concentrations and efficient post-thaw washing to remove CPA are also desirable. The present review focuses on the current strategies and important aspects of adult stem cell banking for clinical applications. These include current good manufacturing practices (cGMPs),animal protein free freezing solutions,cryoprotectants,freezing & thawing protocols,viability assays,packaging and distribution. The importance and benefits of banking clinical grade adult stem cells are also discussed. View Publication

SH3 and multiple ankyrin repeat domains 3 (SHANK3) haploinsufficiency is causative for the neurological features of Phelan-McDermid syndrome (PMDS),including a high risk of autism spectrum disorder (ASD). We used unbiased,quantitative proteomics to identify changes in the phosphoproteome of Shank3-deficient neurons. Down-regulation of protein kinase B (PKB/Akt)-mammalian target of rapamycin complex 1 (mTORC1) signaling resulted from enhanced phosphorylation and activation of serine/threonine protein phosphatase 2A (PP2A) regulatory subunit,B56β,due to increased steady-state levels of its kinase,Cdc2-like kinase 2 (CLK2). Pharmacological and genetic activation of Akt or inhibition of CLK2 relieved synaptic deficits in Shank3-deficient and PMDS patient-derived neurons. CLK2 inhibition also restored normal sociability in a Shank3-deficient mouse model. Our study thereby provides a novel mechanistic and potentially therapeutic understanding of deregulated signaling downstream of Shank3 deficiency. View Publication

We developed a real-time copy number polymerase chain reaction assay for deletions on chromosome 20q (del20q),screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders,and identified 19 patients with del20q (2.9%),of which 14 (74%) were also positive for JAK2-V617F. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F,we performed colony assays in methylcellulose,picked individual burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte (CFU-G) colonies,and genotyped each colony individually for del20q and JAK2-V617F. In 2 of 9 patients,we found that some colonies with del20q carried only wild-type JAK2,whereas other del20q colonies were JAK2-V617F positive,indicating that del20q occurred before the acquisition of JAK2-V617F. However,in colonies from 3 of 9 patients,we observed the opposite order of events. The lack of a strict temporal order of occurrence makes it doubtful that del20q represents a predisposing event for JAK2-V617F. In 2 patients with JAK2-V617F and 1 patient with MPL-W515L,microsatellite analysis revealed that del20q affected chromosomes of different parental origin and/or 9pLOH occurred at least twice. The fact that rare somatic events,such as del20q or 9pLOH,occurred more than once in subclones from the same patients suggests that the myeloproliferative disorder clone carries a predisposition to acquiring such genetic alterations. View Publication

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion,differentiation,migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin,which both localize to stem cell niches in vivo. This matrix allows clonal derivation,clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes. View Publication

Clinical observations suggest that in chronic myelogenous leukemia (CML),the Philadelphia chromosome (Ph+) clone has a growth advantage over normal hematopoiesis. Patients with CML have high levels of neutrophil elastase,which has recently been shown to antagonize the action of granulocyte-colony-stimulating factor (G-CSF) and other growth factors. We therefore compared the effect of elastase on the growth of normal and CML progenitor cells. In 10-day suspension cultures of normal or CML CD34+ cells supplemented with G-CSF,stem cell factor (SCF),and granulocyte macrophage-colony-stimulating factor (GM-CSF),CML cells had diminished sensitivity to the growth inhibitory effect of elastase. When equal numbers of CML and normal CD34+ cells were cocultured for 10 days,there was no change in the relative proportions of normal and leukemic cells (measured by fluorescence in situ hybridization [FISH] or flow cytometry). However,when elastase was added,CML cells predominated at the end of the culture period (78% vs 22% with 1 microg/mL and 80% vs 20% with 5 microg/mL elastase). CML neutrophils substituted effectively for elastase in suppressing the proliferation of normal CD34+ cells,but this effect was abrogated by serine protease inhibitors. These results suggest that elastase overproduction by the leukemic clone can change the growth environment by digesting growth factors,thereby giving advantage to Ph+ hematopoiesis. View Publication