搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

To test the hypothesis that aging has negative effects on stem-cell homing and engraftment,young or old C57BL/6 bone marrow (BM) cells were injected,using a limiting-dilution,competitive transplantation method,into old or young Ly5 congenic mice. Numbers of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) recovered from BM or spleen were measured and compared with the numbers initially transplanted. Although the frequency of marrow competitive repopulation units (CRUs) increased approximately 2-fold from 2 months to 2 years of age,the BM homing efficiency of old CRUs was approximately 3-fold lower than that of young CRUs. Surprisingly,the overall size of individual stem-cell clones generated in recipients receiving a single CRU was not affected by donor age. However,the increased ages of HSC donors and HSC transplant recipients caused marked skewing of the pattern of engraftment toward the myeloid lineage,indicating that HSC-intrinsic and HSC-extrinsic (microenvironmental) age-related changes favor myelopoiesis. This correlated with changes after transplantation in the rate of recovery of circulating leukocytes,erythrocytes,and platelets. Recovery of the latter was especially blunted in aged recipients. Collectively,these findings may have implications for clinical HSC transplantation in which older persons increasingly serve as donors for elderly patients. View Publication

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells,we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells,whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover,long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage,strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether,these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors,and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis. View Publication

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment,proliferation,and differentiation. Given its critical functions,c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay,we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic,functionality of binding was shown. The mimic decreased c-Myb expression,and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest,the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally,in studies using normal human CD34(+) cells,we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation,and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate,these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis. View Publication

Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a congenital deficiency of α-L-iduronidase,leading to lysosomal storage of glycosaminoglycans that is ultimately fatal following an insidious onset after birth. Hematopoietic cell transplantation (HCT) is a life-saving measure in MPS IH. However,because a suitable hematopoietic donor is not found for everyone,because HCT is associated with significant morbidity and mortality,and because there is no known benefit of immune reaction between the host and the donor cells in MPS IH,gene-corrected autologous stem cells may be the ideal graft for HCT. Thus,we generated induced pluripotent stem cells from 2 patients with MPS IH (MPS-iPS cells). We found that α-L-iduronidase was not required for stem cell renewal,and that MPS-iPS cells showed lysosomal storage characteristic of MPS IH and could be differentiated to both hematopoietic and nonhematopoietic cells. The specific epigenetic profile associated with de-differentiation of MPS IH fibroblasts into MPS-iPS cells was maintained when MPS-iPS cells are gene-corrected with virally delivered α-L-iduronidase. These data underscore the potential of MPS-iPS cells to generate autologous hematopoietic grafts devoid of immunologic complications of allogeneic transplantation,as well as generating nonhematopoietic cells with the potential to treat anatomical sites not fully corrected with HCT. View Publication

By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs),we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here,we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction,human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development,it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary,a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells. View Publication

ABSTRACTBackgroundIdiopathic pulmonary fibrosis (IPF) is a lethal disease with an unknown etiology and complex pathophysiology that are not fully understood. The disease involves intricate cellular interplay,particularly among various immune cells. Currently,there is no treatment capable of reversing the fibrotic process or aiding lung regeneration. Hepatocyte growth factor (HGF) has demonstrated antifibrotic properties,whereas the adoptive transfer of modified T cells is a well‐established treatment for various malignancies. We aimed to understand the dynamics of T cells in the progression of lung fibrosis and to study the therapeutic benefit of adoptive T cell transfer in a bleomycin‐injured mouse lung (BLM) model.MethodsT cells were isolated from the spleen of naïve mice and transfected in vitro with mouse HGF plasmid and were administered intratracheally to the mice lungs 7 days post‐bleomycin injury to the lung. Lung tissue and bronchoalveolar lavage were collected and analyzed using flow cytometry,histology,qRT‐PCR,ELISA,and hydroxyproline assay.ResultsOur findings demonstrate the successful T cell therapy of bleomycin‐induced lung injury through the adoptive transfer of HGF‐transfected T cells in mice. This treatment resulted in decreased collagen deposition and a balancing of immune cell exhaustion and cytokine homeostasis compared with untreated controls. In vitro testing showed enhanced apoptosis in myofibroblasts induced by HGF‐overexpressing T cells.ConclusionsTaken together,our data highlight the great potential of adoptive T cell transfer as an emerging therapy to counteract lung fibrosis. This study explores the potential of T cells as a therapeutic strategy against idiopathic pulmonary fibrosis (IPF),a progressive lung disease for which there is currently no treatment to reverse fibrosis or restore normal lung function. To investigate an innovative approach using adoptive T cell transfer,T cells isolated from healthy mice were genetically modified to carry a plasmid containing hepatocyte growth factor (HGF). The modified cells were delivered directly into the airways of mice with bleomycin‐induced lung fibrosis. The results showed a significant reduction in fibrotic scarring,improved immune regulation,and increased apoptosis of pathogenic myofibroblasts. These results highlight the potential of HGF‐engineered T cells as a promising therapeutic approach to combat IPF. View Publication

We examined the stem cell compartment of patients with acquired aplastic anemia (AA) using the long-term culture-initiating cell assay (LTC-IC),in parallel with measurements of CD34+ cells and mature hematopoietic progenitors. Secondary colonies from cells surviving 5 weeks of long-term bone marrow culture (LTBMC) were determined for the peripheral blood (PB) of 68 AA patients and 13 normal controls and for BM of 49 AA patients and 14 controls; because of low cell numbers,formal limiting dilution analysis could only be performed in 10 patients. The relationship of cell input in LTBMC and the output of secondary colonies was linear,allowing quantification of LTC-IC number from bulk cultures. Secondary colony formation was markedly abnormal in severe AA. In contrast to 7.8 colony-forming cells (CFC)/10(5) mononuclear cells in normal BM and 0.14 CFC/10(5) normal PB mononuclear cells,patients with severe disease showed 0.024 CFC/10(5) in BM and 0.0068 CFC/10(5) in PB. Under limiting dilution conditions,patients' cells also showed markedly lower colony-forming ability. In contrast to 4.3 +/- 1 colonies/normal LTC-IC,we obtained only 1.27 +/- 0.09 and 2.0 +/- 0.35 colonies from BM of acute and recovered cases,respectively. These values were used to extrapolate LTC-IC numbers from secondary colony formation in suspension cultures. In PB,calculated LTC-IC were decreased 7.4-fold in new and relapsed severe AA and 2.8-fold in recovered AA. In BM,LTC-IC were decreased 10-fold in new and relapsed AA and sixfold in recovered cases. Compared with measurements obtained on presentation,LTC-IC were lower in post-treatment samples from patients who had failed to recover after intensive immunosuppression and relatively higher in cases at relapse. In recovered patients,LTC-IC number increased but remained below the normal range in 20 of 25. In patients studied serially for 3 to 12 months after treatment,LTC-IC numbers remained stable but low. LTC-IC number correlated with concurrently determined CD34+ cell number and primary hematopoietic colony formation. These results indicate that stem cell numbers,as quantitated by the LTC-IC assay,are markedly diminished in number in all severe AA. Additionally,the function of the stem cell or the stem cell compartment in AA is also abnormal,as inferred from the low clonogenic potential in secondary colony assays. Early hematologic improvement in some patients occurs without increasing numbers of LTC-IC,and a minority of recovered cases show apparent repopulation of the LTC-IC compartment years after treatment. View Publication

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted,self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani,K. Hobson,and M. S. Rao,1997,Dev. Biol. 186,202-223; M. S. Rao and M. Mayer-Proschel,1997,Dev. Biol. 188,48-63; M. Mayer-Proschel,A. J. Kalyani,T. Mujtaba,and M. S. Rao,1997,Neuron 19,773-785). We now show that cells identical to rat NEPs,NRPs,and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs,respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive,undergo self-renewal in defined medium,and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus,lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells. View Publication

Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors,Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However,several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently,it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However,NPCs express Sox2 endogenously,possibly facilitating reprogramming in the absence of exogenous Sox2. In this study,we identified a small-molecule combination,BIX-01294 and BayK8644,that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts,which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors,such as Sox2,and improve reprogramming efficiency. View Publication

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse,but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed textless10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However,the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses textless20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells,mES cells lacking H2AX,a histone protein involved in the DNA damage response,were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining,H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs),an increase in dsb rejoining rate,and a decrease in Ku70/80. Unlike mouse ES,human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells,they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM,and they add to the growing list of differences in the way rodent and human cells deal with DNA damage. View Publication

Neural cells derived from induced pluripotent stem cells (iPSCs) have the potential for autologous cell therapies in treating patients with severe neurological disorders or injury. However,further study of efficacy and safety are needed in large animal preclinical models that have similar neural anatomy and physiology to humans such as the pig. The pig model for pluripotent stem cell therapy has been made possible for the first time with the development of pig iPSCs (piPSCs) capable of in vitro and in vivo differentiation into tissues of all three germ layers. Still,the question remains if piPSCs are capable of undergoing robust neural differentiation using a system similar to those being used with human iPSCs. In this study,we generated a new line of piPSCs from fibroblast cells that expressed pluripotency markers and were capable of embryoid body differentiation into all three germ layers. piPSCs demonstrated robust neural differentiation forming βIII-TUB/MAP2+ neurons,GFAP+ astrocytes,and O4+ oligodendrocytes and demonstrated strong upregulation of neural cell genes representative of all three major neural lineages of the central nervous system. In the presence of motor neuron signaling factors,piPSC-derived neurons showed expression of transcription factors associated with motor neuron differentiation (HB9 and ISLET1). Our findings demonstrate that SSEA4 expression is required for piPSCs to differentiate into neurons,astrocytes,and oligodendrocytes and furthermore develop specific neuronal subtypes. This indicates that the pigs can fill the need for a powerful model to study autologous neural iPSC therapies in a system similar to humans. View Publication

In this study,we examined the effects of cryoprotectant,freezing and thawing,and adenovirus (Adv) transduction on the viability,transgene expression,phenotype,and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh,cryopreserved,and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further,cryopreservation,storage,and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary,cryopreservation,storage,and thawing had no significant effect on DC viability,function,and transgene expression by Adv-transduced DCs. View Publication